Sublingual fentanyl spray

ABSTRACT

The present invention is directed to sublingual formulations containing fentanyl, a pharmaceutically acceptable sale thereof, or derivative thereof, suitable for administration to a patient, and methods for treatment with the formulations.

This application is a continuation of U.S. patent application Ser. No.12/221,333 filed Aug. 1, 2008 (now U.S. Pat. No. 8,486,973 issued Jul.16, 2013), which claims the benefit of U.S. Provisional Application Nos.60/963,076, filed on Aug. 2, 2007 and 60/963,253 filed Aug. 3, 2007; thedisclosures of which are hereby incorporated by reference in theirentireties.

FIELD OF THE INVENTION

The invention is directed to sublingual formulations containingfentanyl, a pharmaceutically acceptable salt thereof, or derivativethereof, suitable for administration to humans, and methods fortreatment with the sublingual formulations.

BACKGROUND OF THE INVENTION

Fentanyl is a μ-opioid receptor agonist with analgesic potencyapproximately 80-100 times that of morphine. In clinical settings,fentanyl exerts its principal pharmacologic effects on the centralnervous system. Its primary actions are analgesic and sedation.

The analgesic effects of fentanyl are related to the blood level of thedrug. In general, the minimum effective concentration and theconcentration at which toxicity occurs rise with increasing tolerance toany and all opioids. The rate of development of tolerance may varywidely among individuals. All opioid mu-receptor agonists, includingfentanyl, produce dose dependent respiratory depression. The risk ofrespiratory depression is typically less in patients receiving chronicopioid therapy who develop tolerance to respiratory depression and otheropioid effects. Serious or fatal respiratory depression can occur, evenat recommended doses, in vulnerable individuals.

Orally administered fentanyl is subject to first pass effect metabolismas upwards of 50% or more of orally administered fentanyl is notabsorbed. Other forms of delivery such a parenteral, buccal, andtransdermal have been utilized to decrease or avoid this first passeffect for fentanyl.

Fentanyl is currently available in injectable form, as a lozenge (e.g.Actiq®; fentanyl citrate; Actiq is a registered trademark of Anesta,LLC), and as a transdermal patch (e.g. Duragesic® 25, 50, 75, and 100 μgof fentanyl per hour; Duragesic is a registered trademark of Johnson &Johnson Corporation). Duragesic® provides continuous systemic deliveryof fentanyl for approximately 72 hours. Duragesic® is indicated in themanagement of chronic pain in patients requiring continuous opioidanalgesia for pain that is not optimally managed with lesser means suchas acetaminophen-opioid combinations, non-steroidal analgesics, or prn(as needed) dosing with short-acting opioids. Duragesic® is typicallynot suitable for patients experiencing acute pain due to the delay inabsorption of the fentanyl through the patch, or postoperative painbecause serious or life-threatening hypoventilation could result.

Actiq® is a solid formulation of fentanyl citrate, intended for oraltransmucosal administration. Actiq® is a lozenge attached to a handlesimilar in shape to a lollipop. The handle is purportedly to allow theActiq® unit to be removed from the mouth if signs of excessive opioideffects appear during administration. Actiq® is indicated for themanagement for breakthrough cancer pain in patients with malignancieswho are already receiving and who are tolerant to opioid therapy fortheir underlying persistent cancer pain. Actiq® is contraindicated inthe management of acute or postoperative pain.

Sublingual tablets and lozenges (e.g., Actiq®) which may be used foracute pain or breakthrough pain have certain disadvantages. Adisadvantage, amongst others, is that after intake the active agent inthese pharmaceutical agents must first be released and dispersed priorto being available for resorption in dissolved form. In addition, theabsorption pharmacokinetics of fentanyl from Actiq® may vary dependingon the fraction of the dose that is absorbed through the oral mucosa andthe fraction swallowed. Further, certain lozenges may be in the form ofa candy which require medical supervision and may be sociallyquestionable.

There exists a need in art for a sublingual formulation includingfentanyl, a pharmaceutically acceptable salt thereof, or derivativethereof, which is suitable for effective pain management.

SUMMARY AND OBJECTS OF THE INVENTION

It is an object of the invention to provide a fentanyl formulationsuitable for sublingual administration for effective pain management.

It is an object of certain embodiments of the invention to providemethods and compositions capable of rapidly inducing a state ofsedation, analgesia, and/or anesthesia.

It is a further object of certain embodiments of the invention toprovide methods and compositions for fentanyl administration whichminimize the underdosing and/or overdosing of a patient in need offentanyl therapy.

It is a further object of certain embodiments of the invention toprovide methods and compositions suitable for the treatment ofbreakthrough pain in patients receiving chronic pain treatment.

It is a further object of certain embodiments of the present inventionto provide a method for sublingual administration of fentanyl, apharmaceutically acceptable salt thereof, or derivative thereof, in acontrolled amount for the treatment of pain.

It is a further object of certain embodiments of the present inventionto provide a dosage form of an opioid analgesic which can beadministered sublingually in a manner which will cause substantialsublingual absorption without substantial risk of the dose passing intothe lungs of the recipient.

The above-mentioned objects and others are achieved by virtue of thepresent invention, which is directed in part to a method forsublingually administering fentanyl, a pharmaceutically acceptable saltthereof, or derivative thereof, to provide fast-acting relief in aformulation in which a substantial portion of the fentanyl, apharmaceutically acceptable salt thereof, or derivative thereof will notbe passed into the lungs of the patient.

In certain embodiments the present invention is directed to a sublingualfentanyl formulation comprising discrete liquid droplets comprising aneffective amount of fentanyl, a pharmaceutically acceptable saltthereof, or derivative thereof, said droplets having a mean diameter ofat least about 10 microns, preferably at least about 20 microns, morepreferably a mean diameter of from about 20 to about 200 microns.

In certain embodiments, the present invention is directed to asublingual fentanyl formulation comprising discrete liquid droplets offentanyl, a pharmaceutically acceptable salt thereof, or derivativethereof; in a pharmaceutically acceptable liquid carrier; said dropletshaving a size distribution of from about 5 microns to about 500 microns,preferably from about 10 microns to about 200 microns, preferably fromabout 20 microns to about 100 microns, more preferably from about 30microns to about 70 microns.

In certain preferred embodiments, none of the particles have a diameterwhich would allow the fentanyl, pharmaceutically acceptable saltthereof, or derivative thereof to be delivered to the lung uponsublingual administration.

In certain embodiments, the present invention is directed to a unit doseof a sublingual fentanyl formulation, said unit dose comprising discreteliquid droplets of fentanyl, a pharmaceutically acceptable salt thereof,or derivative thereof; and a pharmaceutically acceptable liquid carrier;said droplets having a mean diameter of at least about 10 microns,preferably at least about 20 microns, more preferably a mean diameter offrom about 20 to about 200 microns.

In certain embodiments, the present invention is directed to a unit doseof a sublingual fentanyl formulation, said unit dose comprising discreteliquid droplets of fentanyl, a pharmaceutically acceptable salt thereof,or derivative thereof; and a pharmaceutically acceptable liquid carrier;said droplets having a size distribution of from about 5 microns toabout 500 microns, preferably from about 10 microns to about 200microns, preferably from about 20 microns to about 100 microns, morepreferably from about 30 microns to about 70 microns.

In certain embodiments, the present invention is directed to a method oftreating pain comprising sublingually administering a liquid sprayformulation in the form of discrete liquid droplets having a meandiameter of at least about 10 microns, preferably at least about 20microns, more preferably a mean diameter of from about 20 to about 200microns, to a human patient experiencing pain, said liquid sprayformulation comprising an effective amount of fentanyl, apharmaceutically acceptable salt thereof, or derivative thereof,dispersed in a pharmaceutically acceptable liquid carrier.

In certain embodiments, the present invention is directed to a method oftreating pain comprising sublingually administering a liquid sprayformulation in the form of discrete liquid droplets having a sizedistribution of from about 5 microns to about 500 microns, preferablyfrom about 10 microns to about 200 microns, preferably from about 20microns to about 100 microns, more preferably from about 30 microns toabout 70 microns to a human patient experiencing pain; said liquid sprayformulation comprising an effective amount of fentanyl, apharmaceutically acceptable salt thereof, or derivative thereof,dispersed in a pharmaceutically acceptable liquid carrier.

In certain embodiments, the present invention is directed to a devicewhich includes a reservoir containing a unit dose of a liquidformulation comprising an effective amount of fentanyl, apharmaceutically acceptable salt thereof, or derivative thereof in apharmaceutically acceptable liquid carrier; the device having anactuator which when actuated delivers the unit dose of the liquidformulation in the form of liquid droplets having a mean diameter of atleast about 10 microns, preferably at least about 20 microns, morepreferably a mean diameter of from about 20 to about 200 microns.Preferably, the device delivers a therapeutically effective dose of theliquid formulation in the form of liquid droplets having a sizedistribution of from about 5 microns to about 500 microns, preferablyfrom about 10 microns to about 200 microns, preferably from about 20microns to about 100 microns, more preferably from about 30 microns toabout 70 microns.

In certain embodiments, the present invention is directed to amulti-dose device which includes a reservoir containing a liquidformulation comprising fentanyl, a pharmaceutically acceptable saltthereof, or derivative thereof in a pharmaceutically acceptable liquidcarrier; the device having an actuator which when actuated delivers atherapeutically effective dose of the liquid formulation in the form ofliquid droplets having a mean diameter of at least about 10 microns,preferably at least about 20 microns, more preferably a mean diameter offrom about 20 to about 200 microns. Preferably, the device delivers atherapeutically effective dose of the liquid formulation in the form ofliquid droplets having a size distribution of from about 5 microns toabout 500 microns, preferably from about 10 microns to about 200microns, preferably from about 20 microns to about 100 microns, morepreferably from about 30 microns to about 70 microns.

In certain embodiments, the present invention is directed to a method oftreating pain comprising utilizing a spray device which includes areservoir including a liquid formulation comprising fentanyl, apharmaceutically acceptable salt thereof, or derivative thereof in apharmaceutically acceptable liquid carrier; and an actuator which uponactuation delivers a therapeutically effective amount of liquid dropletsto be sprayed from the device having a mean diameter of at least about10 microns, preferably at least about 20 microns, more preferably a meandiameter of from about 20 to about 200 microns.

In certain embodiments, the present invention is directed to a method oftreating pain comprising utilizing a spray device which includes areservoir including a liquid formulation comprising fentanyl, apharmaceutically acceptable salt thereof, or derivative thereof; and apharmaceutically acceptable liquid carrier; and an actuator which uponactuation delivers a therapeutically effective amount of liquid dropletshaving a size distribution of from about 5 microns to about 500 microns,preferably from about 10 microns to about 200 microns, preferably fromabout 20 microns to about 100 microns, more preferably from about 30microns to about 70 microns.

In certain embodiments, the present invention is directed to a method oftreating breakthrough pain comprising sublingually administering aliquid spray formulation comprising an effective amount of fentanyl, apharmaceutically acceptable salt thereof, or derivative thereof,dispersed in a pharmaceutically acceptable liquid carrier to a humanpatient to treat breakthrough pain experienced by said human patient.

In certain embodiments, the present invention is directed to a method oftreating breakthrough pain comprising sublingually administering aliquid spray formulation comprising an effective amount of fentanyl, apharmaceutically acceptable salt thereof, or derivative thereof,dispersed in a pharmaceutically acceptable liquid carrier to a humanpatient who is receiving chronic pain treatment, and is experiencingbreakthrough pain.

In certain embodiments, the present invention is directed to a method ofreducing patient to patient variability for the treatment ofbreakthrough pain, comprising sublingually administering to a humanpatient experiencing breakthrough pain a dose of fentanyl in a liquidspray formulation comprising fentanyl, a pharmaceutically acceptablesalt thereof, or derivative thereof, and a pharmaceutically acceptableliquid carrier said liquid spray formulation being administered asdiscrete liquid droplets having a mean diameter of at least about 10microns, preferably at least about 20 microns, more preferably a meandiameter of from about 20 to about 200 microns.

In certain embodiments, the present invention is directed to a method ofreducing patient to patient variability for the treatment ofbreakthrough pain, comprising sublingually administering to a humanpatient experiencing breakthrough pain a dose of fentanyl in a liquidspray formulation comprising fentanyl, a pharmaceutically acceptablesalt thereof, or derivative thereof, and a pharmaceutically acceptableliquid carrier said liquid spray formulation being administered asdiscrete liquid droplets having a size distribution of from about 5microns to about 500 microns, preferably from about 10 microns to about200 microns.

In certain preferred embodiments, the liquid spray formulation furtherincludes a pharmaceutically acceptable solvent. Preferably thepharmaceutically acceptable solvent is an organic solvent which isincluded in an amount suitable for dissolving the fentanyl, apharmaceutically acceptable salt thereof, or derivative thereof.

In certain preferred embodiments the formulations of the presentinvention provide a mean time to maximum plasma concentration (T_(max))of fentanyl at from about 5 minutes to about 120 minutes, aftersublingual administration to humans.

In certain preferred embodiments the formulations of the presentinvention provide a mean maximum plasma concentration (C_(max)) offentanyl of about 127 pg/ml to about 213 pg/ml per 100 μg fentanyl aftersublingual administration to humans.

In certain preferred embodiments of the present invention theformulations of the present invention do not include a propellant.

In certain embodiments, the formulations of the present invention aresuitable for transmucosal administration, including, for example, buccaladministration.

In certain embodiments, the present invention is further directed to amethod of transmucosally administering fentanyl, a pharmaceuticallyacceptable salt thereof, or derivative thereof, to a human to providefast-acting relief in a formulation in which a substantial portion ofthe fentanyl, a pharmaceutically acceptable salt thereof, or derivativethereof will not be passed into the lungs of the patient. In certainpreferred embodiments, the transmucosal area is the buccal area of ahuman.

In certain embodiments, the present invention is further directed to theuse of a formulation as defined above for the manufacture of amedicament for use as an analgesic, for the treatment of acute painand/or breakthrough pain, as an anesthetic premedication, for theinduction of anesthesia, as a sedative and/or for the treatment ofanxiety.

The invention is also directed to a sublingual fentanyl formulationcomprising discrete liquid droplets of an effective amount of fentanyl,a pharmaceutically acceptable salt thereof, or derivative thereof; in apharmaceutically acceptable liquid carrier; said droplets having a meandiameter of at least about 10 microns, and upon administration to ahuman patient, at least about 90% of the discrete liquid droplets have amean diameter equal or greater than about 9 μm. In other embodiments,not more than about 5% of the discrete liquid droplets have a meandiameter less than 9 μm. In still other embodiments, the formulationprovides a respirable dose of not more than about 5% of the totalfentanyl dose contained.

The invention is also directed to a method of treating pain comprisingsublingually administering a liquid spray formulation in the form ofdiscrete liquid droplets having a mean diameter of at least about 10microns to a human patient experiencing pain and at least about 90% ofthe discrete liquid droplets have a mean diameter equal or greater thanabout 9 μm upon administration to a human patient, said liquid sprayformulation comprising an effective amount of fentanyl, apharmaceutically acceptable salt thereof, or derivative thereof,dispersed in a pharmaceutically acceptable liquid carrier. In certainother embodiments, not more than about 5% of the discrete liquiddroplets have a mean diameter less than 9 μm. In other embodiments, theformulation provides a respirable dose of not more than about 5% of thetotal fentanyl dose contained.

The invention is also directed to a unit dose or bi-dose device forsublingual administration of a drug comprising:

a reservoir containing a unit dose or a bi-dose of a liquid formulationcomprising an effective amount of fentanyl, a pharmaceuticallyacceptable salt thereof, or derivative thereof in a pharmaceuticallyacceptable liquid carrier; and

the device having an actuator which when actuated delivers the unit doseof the liquid formulation in the form of liquid droplets having a meandiameter of at least about 10 microns, and wherein upon administrationto a human patient, at least about 90% of the discrete liquid dropletshave a mean diameter equal or greater than about 9 μm.

In other embodiments, not more than about 5% of the discrete liquiddroplets have a mean diameter less than 9 μm. In still otherembodiments, the formulation provides a respirable dose of not more thanabout 5% of the total fentanyl dose contained.

In accordance with the above objects, the invention is also directed toa sublingual spray formulation comprising an effective amount offentanyl and at least one pharmaceutically acceptable excipient, theformulation providing a mean Tmax of about 1.28+/−0.60 hours when a doseis administered sublingually to humans. In certain other embodiments,the sublingual formulation has a concentration of fentanyl from about 1mg/mL to about 8 mg/mL. In certain preferred embodiments, theconcentration of fentanyl is about 1 mg/mL, about 2 mg/mL, about 4mg/mL, about 6 mg/mL or about 8 mg/mL.

In accordance with certain of the above objections, the invention isalso directed to a sublingual formulation exhibiting a mean Cmax ofabout 0.813 ng/ml+/−0.252 based on a sublingual dose of about 400 mcgfentanyl when administered to humans.

In certain other embodiments, the sublingual formulation provides adosage amount of fentanyl selected from the group consisting of about100 mcg, about 200 mcg, about 600 mcg and about 800 mcg, and provides amean Cmax which is substantially dose proportional to the sublingualformulation containing 400 mcg fentanyl dosage amount, when administeredto humans.

In still other embodiments, the sublingual formulation provides asubstantially dose proportional mean Cmax based on a mean Cmax of about0.813 ng/ml+/−0.252 for a 400 mcg fentanyl dose when administered tohumans.

The present invention also provides a sublingual formulation whichprovides a mean Tmax when administered to humans selected from the groupconsisting of: about 1.12 hours when the formulation provides a 100 mcgdose, about 1.04 hours when the formulation provides a 200 mcg dose,about 0.97 hours when the formulation provides a 400 mcg dose, about0.987 hours when the formulation provides a 600 mcg dose, and about 1.06hours when the formulation provides a 800 mcg dose.

In accordance with the above objects, it is a further object of theinvention to provide a sublingual fentanyl formulation which provides aplasma concentration after administration to humans selected from thegroup consisting of: about 60% of the mean Cmax in about 10 minutes,about 86% of the mean Cmax by about 20 minutes and a combinationthereof.

In other embodiments, the invention is directed to a sublingual fentanylformulation that when administered to humans provides a plasmaconcentration that is greater than about 80% of the mean Cmax for about2 hours.

In still other embodiments, the sublingual formulation comprises 400 mcgof fentanyl, providing one or more mean pharmacokinetic values selectedfrom the group consisting of: AUClast 4.863+/−1.70821 hr*ng/mL, AUCinf5.761+/−1.916 hr*ng/mL, and AUCextrap 10.26+/−5.66%, when administeredto humans.

In even still further embodiments of the invention, the sublingualfentanyl formulation provides a dosage amount of fentanyl whenadministered to humans which is substantially dose proportional to thedosage which contains about 400 mcg fentanyl selected from the groupconsisting of about 100 mcg, about 200 mcg, about 600 mcg, about 800meg, and provides one or more pharmacokinetic values selected from thegroup consisting of: mean AUC_(last), mean AUC_(inf), and meanAUC_(extrap).

In still further embodiments of the invention, the sublingual fentanylformulation provides a substantially dose proportional mean AUClastbased on a mean AUClast of about 4.863+/−1.70821 hr*ng/mL for a 400 mcgfentanyl dose when administered to humans.

It is also an object of the invention to a provide a sublingualformulation comprising a 400 mcg dose of fentanyl, providing a geometricmean ln(Cmax) of about 0.7865 ng/ml when a dose is administered tohumans.

In still other embodiments, the sublingual formulation comprises a 400mcg dose of fentanyl, providing a mean F(AUClast) of about 0.721+/−0.199ng/mL when a dose is administered to humans.

In other embodiments, the invention is directed to a sublingualformulation comprising a 400 mcg dose of fentanyl that when administeredto humans, provides a mean F (bioavailability) selected from the groupconsisting of: about 71%+/−16%, 0.721+/−0.199 based on AUClast and about0.756+/−0.212 based on AUCinf, or combinations thereof.

In accordance with the above objects, the invention is further directedto a sublingual fentanyl spray formulation that further comprises water.In other embodiments, the formulations further comprise dehydratedalcohol.

It is also an object of the invention to provide a method of treatingpain comprising administering to a patient in need thereof a sublingualspray formulation comprising an effective amount of fentanyl and atleast one pharmaceutically acceptable excipient, the formulationproviding a mean Tmax of about 1.28+/−0.60 hours when a dose isadministered sublingually to humans.

In accordance with the above objects, the invention is also directed toa method of treatment wherein the sublingual formulation provides asubstantially dose proportional mean Cmax based on a mean Cmax of about0.813 ng/ml+/−0.252 for a 400 mcg fentanyl dose when administered tohumans.

In yet further embodiments, the invention is also directed to a methodof treatment wherein the sublingual formulation provides a substantiallydose proportional mean AUClast based on a mean AUClast of about4.863+/−1.70821 hr*ng/mL for a 400 mcg fentanyl dose when administeredto humans.

It is a further object of the invention to provide a method ofmanufacturing a sublingual spray formulation comprising an effectiveamount of fentanyl and at least one pharmaceutically acceptableexcipient comprising: admixing fentanyl, purified water and dehydratedalcohol and placing the mixture into a sublingual delivery device;wherein the formulation provides a mean Tmax of about 1.28+/−0.60 hourswhen a dose is administered sublingually to humans and upon deliveryprovides particles having a mean aerodynamic particle size of at leastabout 10 microns.

Many patients with e.g., cancer, typically continue to experiencemoderate to severe pain despite chronic analgesic therapy and this canoccur as intermittent breakthrough pain, often due to increases in apatient's activity level. Attempts to counteract this type of pain byincreasing the dose of long-acting formulations of analgesics oftenproduce slow onset of analgesia and unwanted side-effects of sedation,constipation or nausea and vomiting. However, in certain embodiments thepresent invention is directed to a formulation which preferably providesa rapidly acting, potent analgesic which reduces the pain, such asbreakthrough pain, for the required time and then preferably wears offfairly quickly thereby minimizing the side-effects of the fentanyl, apharmaceutically acceptable salt thereof, or derivative thereof.

For purposes of the present invention, derivatives of fentanyl includesufentanil, carfentanil, lofentanil, alfentanil, or the like.

For purposes of the present invention, “breakthrough pain” refers to apain that exceeds a threshold in a patient which causes cognizablediscomfort wherein the pain experienced by the patient is otherwisetypically controlled e.g., by chronic analgesic therapy, and tolerated.For example, pain related to medical illnesses, such as cancer,typically fluctuates, and patients often report the experience ofcognizable discomfort (e.g., breakthrough pain). Typically breakthroughpain is currently treated with immediate release oral dosage forms whichmay take up to about 45 minutes or longer for the drug to be absorbedand may result in a delay of the relief of breakthrough pain, as opposedto a liquid spray formulation of the present invention which begins toprovide relief of the breakthrough pain almost immediately afteradministration.

For purposes of the present invention, “chronic pain treatment” refersto a daily or round-the-clock pain treatment. Chronic pain treatment canbe oral, parenteral, transdermal, or other suitable means ofadministration.

For purposes of the present invention, “sublingual” is defined herein asbeneath or concerning the area under the tongue.

For purposes of the present invention the term “sublingualadministration” is defined herein as the therapeutic administration of apharmaceutical composition under the tongue.

For purposes of the present invention an “effective amount” of a drug isan amount effective to demonstrate a desired activity of the drug.According to the instant invention, a therapeutically effective amountof fentanyl, pharmaceutically acceptable salt thereof, or derivativethereof, is an amount effective to treat, e.g., noticeably reduce, painin a patient.

For purposes of the present invention the terms droplets and particlesmay be used interchangeably.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the mean (±S.E.) plasma concentration-time profilesfollowing intravenous administration of Fentanyl of Example 1 (n=3) inthe study of Example 6.

FIG. 2 depicts the mean (±S.E.) plasma concentration-time profilesfollowing sublingual administration of Fentanyl of Example 1 (n=3) inthe study of Example 6.

FIG. 3 depicts the mean (±S.E.) plasma concentration-time profilesfollowing intravenous administration of Fentanyl of Example 2 (n=3) inthe study of Example 6.

FIG. 4 depicts the mean (±S.E.) plasma concentration-time profilesfollowing sublingual administration of Fentanyl of Example 2 (n=3) inthe study of Example 6.

FIG. 5 depicts the mean (±S.E.) plasma concentration-time profilesfollowing intravenous administration of Fentanyl of Example 3 (n=3) inthe study of Example 6.

FIG. 6 depicts the mean (±S.E.) plasma concentration-time profilesfollowing sublingual administration of Fentanyl of Example 3 (n=3) inthe study of Example 6.

FIG. 7 depicts the mean (±S.E.) plasma concentration-time profilesfollowing intravenous administration of Fentanyl of Example 4 (n=3) inthe study of Example 6.

FIG. 8 depicts the mean (±S.E.) plasma concentration-time profilesfollowing sublingual administration of Fentanyl of Example 4 (n=3) inthe study of Example 6.

FIG. 9 depicts the mean (±S.E.) plasma concentration-time profilesfollowing intravenous administration of Fentanyl of Example 5 (n=3) inthe study of Example 6.

FIG. 10 depicts the mean (±S.E.) plasma concentration-time profilesfollowing sublingual administration of Fentanyl of Example 5 (n=3) inthe study of Example 6.

FIG. 11 depicts a graphical summary of the Dv10, Dv50, Dv90, and plumerecords values versus placement distance for vignetting results.

FIG. 12 depicts a graphical summary of Dv10, Dv50, and Dv90 valuesversus placement at 4 cm device to laser beam for exhaust results.

FIG. 13 depicts a graphical summary of Dv10, Dv50, and Dv90 valuesversus placement at 7 cm device to laser beam for exhaust results.

FIG. 14 depicts a graphical summary of device to laser beam placementresults for Dv10, Dv50, and Dv90 values versus distance to device tolaser beam.

FIG. 15 depicts the mean fentanyl concentration-time profiles afteradministration of Fentanyl Sublingual Spray 400 mcg (Treatment A),Actiq® 400 mcg (Treatment B) and Fentanyl Citrate Injection 100 mcg(Treatment C) in the clinical study of Example 19.

FIG. 16 depicts the preliminary pharmacokinetic data in the doseescalation study of Example 20.

FIG. 17 depicts the preliminary pharmacokinetic data in the doseescalation study of Example 20.

FIG. 18 depicts the preliminary pharmacokinetic data in the doseescalation study of Example 20.

DETAILED DESCRIPTION OF THE INVENTION

The simplest and most prevalent administration route for pharmacologicagents is by mouth. To use this method, a pharmacological agent istypically incorporated into a tablet, a capsule, or into a liquid base.Oral administration of a drug is extremely convenient, and for manydrugs, it will continue to be the method of choice.

Absorption of a drug into the bloodstream after swallowing a tablet mayvary from patient to patient. The absorption of the drug is typicallydependent upon the movement from the stomach to the small and largeintestines and the effects of secretions from these organs. Further,with the oral administration of a drug such as fentanyl to a patient, asthe fentanyl enters the patient's bloodstream through the intestines andpasses through the patient's liver before distribution throughout thebody, upwards of fifty percent or more of the fentanyl may be removedfrom the patient's bloodstream. This “first pass effect” results in theoral route of administration being impractical for fentanyl.

Absorption of fentanyl or a pharmaceutically acceptable salt thereofinto the bloodstream following oral administration is significantlyreduced by the first pass effect. Therefore, the oral route ofadministration is impractical for fentanyl. Other forms of delivery sucha parenteral, buccal, and transdermal delivery have been utilized todecrease or avoid this first pass effect for fentanyl. However, theseother forms of delivery have certain disadvantages associated with them.For example, parenteral administration requires injection using asyringe and needle, and may lead to necrosis that can accompany i.m.administration of drugs; Actiq®, a transmucosal fentanyl citrate lozengeformulation requires the patient to constantly suck on the lozenge whichis attached to a handle (similar to a lollipop) in order to obtaineffective pain relief; and Duragesic®, a transdermal fentanyl deliverydevice, is suitable for the management of chronic pain, but is notindicated for acute or breakthrough pain.

The oral cavity offers a simple, painless method of opioid analgesicadministration. Within the oral cavity, there are three generallyrecognized routes of administration of an active agent, namely local,buccal and sublingual.

Local delivery is mainly limited to applications regarding disruptionsoccurring within the oral cavity itself, such as a canker sore.

The buccal mucosa area encompasses the mucosal membranes of the innerlining of the cheeks. The buccal mucosa is however, less permeable thanthe sublingual area. One of the major disadvantages associated withbuccal mucosa delivery of an active agent has been the relatively lowpassage of active agents across the mucosal epithelium, therebyresulting in low agent bioavailability, which translates into asubstantial loss of usable active agent within each dosage.

Sublingual delivery is achieved through the mucosal membranes lining thefloor of the mouth. Because of the high permeability and the rich bloodsupply, transport via the sublingual route results in a rapid onset ofaction, providing a delivery route appropriate for highly permeabledrugs with short delivery period requirements and an infrequent dosingregimen.

The sublingual formulations of the present invention are useful in thetreatment of moderate to severe pain. Preferably the sublingualformulations of the present invention are useful for the treatment ofbreakthrough pain. For example, the formulations of the presentinvention are preferably suitable for a patient receiving chronic paintherapy who experiences breakthrough pain and is in need of acute painrelief.

The sublingual formulations of the present invention may be used toalleviate pain from many causes, including but not limited to shock,limb amputation, severe chemical or thermal burn injury, sprains,ligament tears, fractures, wounds and other tissue injuries, dentalsurgery, procedures and maladies, labor and delivery, during physicaltherapy, post operative pain, radiation poisoning, cancer, acquiredimmunodeficiency syndrome (AIDS), epidural (or peridural) fibrosis, backsurgery and laminectomy, sciatica, painful sickle cell crisis,arthritis, autoimmune disease, intractable bladder pain, and the like.Sublingual administration of the formulations of fentanyl, apharmaceutically acceptable salt thereof, or derivative thereof, of thepresent invention is also preferably amenable to hospice use,particularly hospices that specialize in the care of cancer and AIDSpatients.

In certain preferred embodiments, the sublingual administration offentanyl, a pharmaceutically acceptable salt thereof, or derivativethereof, can relieve or alleviate episodes of acute breakthrough painthat can occur in a chronic pain condition. In a further embodiment,sublingual administration of fentanyl, pharmaceutically acceptable saltthereof, or derivative thereof can be used as an adjunct therapy to aconventional treatment regimen for a chronic pain condition to alleviatebreakthrough pain. In certain embodiments, the invention also provides aformulation for use as an anesthetic premedication, for the induction ofanesthesia, for use as a sedative and/or for the treatment of anxiety.

Sublingual administration of fentanyl, a pharmaceutically acceptablesalt thereof, or derivative thereof, in accordance with the presentinvention may be particularly beneficial in the patient with cancer whois unable to tolerate oral administration because of nausea andvomiting, dysphagia as a result of disease, or parenteral administrationbecause of decreased venous access, emaciation, or coagulation defects.Sublingual administration of fentanyl in accordance with the presentinvention preferably has potential advantages of even greater ease ofuse and rapid onset of pain relief action. Furthermore, becausesublingual venous drainage is systemic rather than portal, hepaticfirst-pass elimination may be avoided. The present invention preferablyprovides therapeutic formulations and methods for solutions of fentanyl,a pharmaceutically acceptable salt thereof, or derivative thereof to bedelivered by sublingual spray pumps.

In certain preferred embodiments, the sublingual administration offentanyl, a pharmaceutically acceptable salt thereof, or derivativethereof, is advantageous over other forms of administration in that itdoes not require injection using a syringe and needle, it avoidsnecrosis that can accompany i.m. administration of drugs, and it avoidsthe need to constantly suck on a lozenge or lollipop. Preferably thesublingual administration of fentanyl, a pharmaceutically acceptablesalt thereof, or derivative thereof, in accordance with the presentinvention is suitable for self administration.

In preferred embodiments certain embodiments, the formulations of thepresent invention are advantageous in that propellants such ashydrofluorocarbon propellants such as volatile chlorofluocarbons (e.g.propellant 12), volatile hydrofluoroalkanes (e.g.1,1,1,2-tetrafluoroethane and 1,1,1,2,3,3,3-heptafluoro-n-propane) andvolatile alkanes (e.g. propane, butane) are not required to deliver thefentanyl, a pharmaceutically acceptable salt thereof, or derivativethereof, sublingually to the patient.

Preferably the formulations of the present invention are delivered asliquid droplets having a mean diameter of at least about 10 microns,preferably at least about 20 microns, more preferably a mean diameter offrom about 20 to about 200 microns. Most preferably the formulations aredelivered as liquid droplets have a size distribution of from about 5microns to about 500 microns, preferably from about 10 microns to about200 microns, preferably from about 20 microns to about 100 microns, morepreferably from about 30 microns to about 70 microns.

Preferably the delivery of the formulation of the present invention tothe sublingual mucosa via spray results in a rapid onset of therapeuticeffect of the fentanyl, a pharmaceutically acceptable salt thereof, orderivative thereof. For example, in certain embodiments the formulationsof the present invention provide a mean time to maximum plasmaconcentration (T_(max)) of fentanyl at from about 5 minutes to about 120minutes, preferably at from about 10 to about 60 minutes, and morepreferably at from about 15 to about 35 minutes after sublingualadministration to humans.

In certain further embodiments the formulations of the present inventionprovide a mean maximum plasma concentration (C_(max)) of fentanyl ofabout 127 pg/ml to about 213 pg/ml per 100 μg fentanyl, preferably about142 pg/ml to about 195 pg/ml per 100 μg fentanyl, more preferably about158 pg/ml to about 177 pg/ml per 100 μg fentanyl after sublingualadministration to humans.

In certain further embodiments the formulations of the present inventionprovide a mean maximum plasma concentration (C_(max)) of fentanyl ofabout 137 pg/ml to about 207 pg/ml based on a 100 μg fentanyl dose,preferably about 154 pg/ml to about 190 pg/ml based on a 100 μg fentanyldose, more preferably about 163 pg/ml to about 181 pg/ml based on a 100μg fentanyl dose after sublingual administration to humans.

In certain further embodiments the formulations of the present inventionprovide a mean maximum plasma concentration (C_(max)) of fentanyl ofabout 566 pg/ml to about 850 pg/ml based on a 400 μg fentanyl dose,preferably about 637 pg/ml to about 779 pg/ml based on a 400 μg fentanyldose, more preferably about 672 pg/ml to about 744 pg/ml based on a 400μg fentanyl dose after sublingual administration to humans.

In certain further embodiments the formulations of the present inventionprovide a mean maximum plasma concentration (C_(max)) of fentanyl ofabout 1016 pg/ml to about 1525 pg/ml based on a 800 μg fentanyl dose,preferably about 1143 pg/ml to about 1398 pg/ml based on a 800 μgfentanyl dose, more preferably about 1206 pg/ml to about 1334 pg/mlbased on a 800 μg fentanyl dose after sublingual administration tohumans.

In certain further embodiments the formulations of the present inventionprovide an area under the plasma concentration time curve to infinity(AUC_(∞)) of fentanyl of about 572 pg·h/ml to about 1273 pg·h/ml per 100μg fentanyl, preferably about 644 pg·h/ml to about 1167 pg·h/ml per 100μg fentanyl, more preferably about 715 pg·h/ml to about 1061 pg·h/ml per100 μg fentanyl after sublingual administration to humans.

In certain further embodiments the formulations of the present inventionprovide an area under the plasma concentration time curve to infinity(AUC_(∞)) of fentanyl of about 654 pg·h/ml to about 982 pg·h/ml based ona 100 μg fentanyl dose, preferably about 736 pg·h/ml to about 900pg·h/ml based on a 100 μg fentanyl dose, more preferably about 777pg·h/ml to about 859 pg·h/ml based on a 100 μg fentanyl dose aftersublingual administration to humans.

In certain further embodiments the formulations of the present inventionprovide an area under the plasma concentration time curve to infinity(AUC_(∞)) of fentanyl of about 3394 pg·h/ml to about 5092 pg·h/ml basedon a 400 μg fentanyl dose, preferably about 3818 pg·h/ml to about 4667pg·h/ml based on a 400 pg fentanyl dose, more preferably about 4030pg·h/ml to about 4455 pg·h/ml based on a 400 μg fentanyl dose aftersublingual administration to humans.

In certain further embodiments the formulations of the present inventionprovide an area under the plasma concentration time curve to infinity(AUC_(∞)) of fentanyl of about 4581 pg·h/ml to about 6873 pg·h/ml basedon a 800 μg fentanyl dose, preferably about 5154 pg·h/ml to about 6300pg·h/ml based on a 800 μg fentanyl dose, more preferably about 5440pg·h/ml to about 6014 pg·h/ml based on a 800 μg fentanyl dose aftersublingual administration to humans.

In certain further embodiments the formulations of the present inventionprovide an area under the plasma concentration time curve from zero tothe time of the last quantifiable plasma concentration (AUC_(T)) offentanyl of about 378 pg·h/ml to about 1067 pg·h/ml per 100 μg fentanyl,preferably about 425 pg·h/ml to about 978 pg·h/ml per 100 μg fentanyl,more preferably about 472 pg·h/ml to about 889 pg·h/ml per 100 μgfentanyl after sublingual administration to humans.

In certain further embodiments the formulations of the present inventionprovide an area under the plasma concentration time curve from zero tothe time of the last quantifiable plasma concentration (AUC_(T)) offentanyl of about 378 pg·h/ml to about 568 pg·h/ml based on a 100 μgfentanyl dose, preferably about 425 pg·h/ml to about 520 pg·h/ml basedon a 100 μg fentanyl dose, more preferably about 448 pg·h/ml to about497 pg·h/ml based on a 100 μg fentanyl dose after sublingualadministration to humans.

In certain further embodiments the formulations of the present inventionprovide an area under the plasma concentration time curve from zero tothe time of the last quantifiable plasma concentration (AUC_(T)) offentanyl of about 2844 pg·h/ml to about 4268 pg·h/ml based on a 400 μgfentanyl dose, preferably about 3200 pg·h/ml to about 3912 pg·h/ml basedon a 400 μg fentanyl dose, more preferably about 3378 pg·h/ml to about3734 pg·h/ml based on a 400 μg fentanyl dose after sublingualadministration to humans.

In certain embodiments, an increase in pH of the formulations increasesabsorption.

In certain other embodiments, an increase in temperature increasesabsorption.

In certain further embodiments the formulations of the present inventionprovide an area under the plasma concentration time curve from zero tothe time of the last quantifiable plasma concentration (AUC_(T)) offentanyl of about 4333 pg·h/ml to about 6501 pg·h/ml based on a 800 μgfentanyl dose, preferably about 4875 pg·h/ml to about 5960 pg·h/ml basedon a 800 μg fentanyl dose, more preferably about 5146 pg·h/ml to about5689 pg·h/ml based on a 800 μg fentanyl dose after sublingualadministration to humans.

Preferably the fentanyl, a pharmaceutically acceptable salt thereof, orderivative thereof, is dissolved in an organic solvent. Examples oforganic solvents that may be used to enhance the solubility of fentanyl,or the pharmaceutically acceptable salt thereof in a carrier such ase.g., water, include for example and without limitation: lower alcohols(e.g. C₁₋₄ alcohols) such as methanol, ethanol, propyl alcohol, or butylalcohol; C₂₋₈ alcohols having two or three hydroxyl groups, preferablyglycerol, propylene glycol or butylene glycol; and polyethylene glycolssuch as PEG₂₀₀ and PEG₄₀₀ and the like. Mixtures of any of theaforementioned solvents may be used. In certain embodiments, the solventis a non-polar hydrocarbon, preferably a C₇₋₁₈ hydrocarbon of a linearor branched configuration, its alcohols, fatty acid esters, andtriglycerides, such as miglyol. In certain preferred embodiments theorganic solvent is ethanol, propylene glycol, polyethylene glycol, orcombination thereof.

Preferably the amount of organic solvent for inclusion in theformulation is at least an amount of organic solvent necessary toadequately solubilize the fentanyl, a pharmaceutically acceptable saltthereof, or derivative thereof, such that the fentanyl remains insolution and does not precipitate out.

In certain embodiments, the organic solvent is included in theformulation in an amount of from about 0% to about 99.9% by weight ofthe formulation, preferably from about 10% to about 80% by weight of theformulation, more preferably from about 20% to about 60% by weight ofthe formulation.

In certain embodiments, the compositions comprise a C₂₋₈ alcohol such aspropylene glycol, or a polyethylene glycol and/or polypropylene glycolof an average molar weight of 200 to 4000, or a mixture thereof, inaddition to the organic solvent described above. The C₂₋₈ alcohol mayact as a cosolvent in combination with the organic solvent. Polyethyleneglycols commercially available as Carbowax® (Carbowax is a registeredtrademark of Union Carbide Corporation; e.g., Carbowax® 300 of a molarweight of 300), can be used.

In certain embodiments, the solvent is a cosolvent which includes any ofthe solvents mentioned herein. In certain preferred embodiments, thecosolvent includes ethanol, propylene glycol, polyethylene glycol,labrosol, labrafil, transcutol, or combination thereof

In certain preferred embodiments, the composition according to theinvention comprises from about 0.0001% to about 20% by weight offentanyl, a pharmaceutically acceptable salt thereof, or derivativethereof; from about 1% to about 99% by weight of organic solvent; andfrom about 0.01% to about 50% by weight of C₂₋₈ alcohol.

In certain preferred embodiments, the composition according to theinvention comprises from about 0.001% to about 15% by weight offentanyl, a pharmaceutically acceptable salt thereof, or derivativethereof; from about 5% to 90% by weight of ethanol; and from about 0.1%to 40% by weight of propylene glycol.

In certain preferred embodiments, the composition according to theinvention comprises from about 0.01% to about 10% by weight of fentanyl,a pharmaceutically acceptable salt thereof, or derivative thereof; fromabout 10% to about 80% by weight of ethanol; and from about 1% to about30% by weight of propylene glycol.

In certain preferred embodiments, the composition according to theinvention comprises from about 0.1% to about 0.8% by weight of fentanyl,a pharmaceutically acceptable salt thereof, or derivative thereof; fromabout 20% to about 60% by weight of ethanol; and from about 4% to about6% by weight of propylene glycol.

In certain preferred embodiments, the composition according to theinvention comprises in a 1 ml volume: from about 100 μg/ml to about 800μg/ml fentanyl base, about 50% ethanol, about 5.2% propylene glycol, andwater qs to 1 ml.

In certain embodiments the fentanyl is employed in the form of apharmaceutically acceptable salt. Examples of suitable salt forms offentanyl for use in accordance with the present invention include forexample and without limitation, the hydrochloride, chloride, sulphate,tartrate, or citrate salt forms. In certain preferred embodiments, thefentanyl is employed as the free base in the formulations of the presentinvention.

In certain preferred embodiments, the fentanyl, pharmaceuticallyacceptable salt thereof, or derivative thereof, will be employed in theformulation at a concentration of from about 0.05 mg/ml to about 15mg/ml, preferably from about 0.1 mg/ml to about 10 mg/ml, morepreferably from about 1 mg/ml to about 8 mg/ml (where weight isexpressed as weight of fentanyl free base).

In certain preferred embodiments, the amount of fentanyl,pharmaceutically acceptable salt thereof, or derivative thereof;delivered per unit dose is about 10 μg to about 10 mg, preferably fromabout 25 μg to about 5 mg, more preferably from about 50 μg to about1600 μg.

In preferred embodiments of the present invention, the formulation is asolution. In certain alternate embodiments, the formulation is asuspension. When the formulation of the present invention is asuspension, it may be necessary to shake the composition prior tospraying.

In certain preferred embodiments, after the fentanyl, a pharmaceuticallyacceptable salt thereof, or derivative thereof, is dissolved in theorganic solvent, the formulation is preferably included into a liquidcarrier for the delivery of the fentanyl, a pharmaceutically acceptablesalt thereof, or derivative thereof, via a spray device.

Pharmaceutically acceptable carriers include but are not limited towater, buffer, saline, buffered saline, dextrose solution, propyleneglycol, polyethylene glycols, miglyol, and the like. In a specificembodiment, a carrier that may be used in the pharmaceutical formulationof the present invention is phosphate buffered saline, or a bufferedsaline. In certain preferred embodiments the carrier is water. Incertain embodiments, the water in the formulation is present in the formof an aqueous buffer. The buffer is preferably adapted to stabilize thepH of the formulation at pH of about 5 to about 12, preferably at pH ofabout 6 to about 10, more preferably from about 8 to about 9.5. Buffersystems for use in accordance with the present invention include forexample and without limitation sodium acetate/acetic acid, ammoniumacetate/disodium edentate, boric acid/sodium hydroxide, orthophosphoricacid/sodium hydroxide, sodium hydrogen carbonate/sodium carbonate,disodium hydrogen orthophosphate/citric acid, and the like.

Other components such as preservatives, antioxidants, surfactants,absorption enhancers, viscosity enhancers or film forming polymers,bulking agents, diluents, coloring agents, flavoring agents, pHmodifiers, sweeteners or taste-masking agents may also be incorporatedinto the composition. Suitable coloring agents include red, black andyellow iron oxides and FD&C dyes such as FD&C Blue No. 2, FD&C Red No.40, and the like. Suitable flavoring agents include mint, raspberry,licorice, orange, lemon, grapefruit, caramel, vanilla, cherry grapeflavors, combinations thereof, and the like. Suitable pH modifiersinclude citric acid, tartaric acid, phosphoric acid, hydrochloric acid,maleic acid, sodium hydroxide, and the like. Suitable sweeteners includeaspartame, acesulfame K, thaumatic, and the like. Suitable taste-maskingagents include sodium bicarbonate, ion-exchange resins, cyclodextrininclusion compounds, adsorbates, and the like.

Absorption enhancers for use in accordance with the present inventioninclude, for example, polysorbates, sorbitan esters, poloxamer blockcopolymers, PEG-35 castor oil, PEG-40 hydrogenated castor oil,caprylocaproyl macrogol-8 glycerides, PEG-8 caprylic/capric glycerides,sodium lauryl sulfate, dioctyl sulfosuccinate, polyethylene laurylether, ethoxydiglycol, propylene glycol mono-di-caprylate, glycerolmonocaprylate, glyceryl fatty acids (C₈-C₁₈) ethoxylated, oleic acid,linoleic acid, glyceryl caprylate/caprate, glyceryl monooleate, glycerylmonolaurate, caprylic/capric triglycerides, ethoxylated nonylphenols,PEG-(8-50) stearates, olive oil PEG-6 esters, triolein PEG-6 esters,lecithin, d-alpha tocopheryl polyethylene glycol 1000 succinate,polycarbonate, sodium glycocholate, sodium taurocholate, cyclodextrins,citric acid, sodium citrate, triacetin, combinations thereof, and thelike. In certain preferred embodiments, the absorption enhancer istriacetin. In certain preferred embodiments wherein an absorptionenhancer is included in the formulation, the absorption enhancer isincluded in an amount of from about 0.001% to about 10% by weight of theformulation, preferably in an amount of about 0.01% to about 5% byweight of the formulation.

Bulking agents for use in accordance with the present invention includefor example, microcrystalline cellulose, mannitol, xylitol, starches andthe like. In certain preferred embodiments, the bulking agent ismannitol. In certain preferred embodiments wherein bulking agent isincluded in the formulation, the bulking agent is included in an amountof from about 0.001% to about 10% by weight of the formulation,preferably in an amount of about 0.01% to about 5% by weight of theformulation.

Film-forming polymers for use in accordance with the present inventionmay serve for decreasing the fineness of the spray, the spraying angleand preferably the spreading by increasing the viscosity of thecomposition. As film-forming polymer, gellan gum, xantham gum,carboxymethyl cellulose, hydroxypropyl cellulose, hydroxyethylcellulose, hydroxypropyl methylcellulose, methylcellulose,ethylcellulose, gelucire, poloxamers, alginic acid, propyleneglycolester, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), PVP/PVAcopolymer, lubrajel, carboxyvinyl polymer, acrylic acid polymers andcopolymers, methacrylic acid, methyl acrylate, ethyl acrylate, methylmethacrylate, ethyl methacrylate, combinations thereof and the like canbe used. In certain embodiments, an increase in the viscosity of thesolution using film-forming polymers or the like provides an increase inthe droplet size when administered from the spray device. The chemistryof the polymer and the molecular weight of the polymer may alsoinfluence the diameter of the droplets.

In certain embodiments, the formulations according to the invention arepreferably packaged as a bulk solution containing multiple doses in apump spray system comprising a sealed container fitted with a meteringpump.

In certain alternate embodiments the formulations according to theinvention are preferably package as a single unit dose solution in asingle unit dose pump spray system comprising a sealed container fittedwith a pump.

Typically a patient is treated by administration sublingually of 1 to 2actuations, from the spray pump. Another advantage of sublingual spraydelivery is the ability to easily titrate patients by 1 or 2 doses asrequired by a single actuation. This is typically not the case withother forms of drug delivery (patches, lozenges, tablets,suppositories).

Pump action sprays are characterized in requiring the application ofexternal pressure for actuation, for example, external manual,mechanical or electrically initiated pressure. This is in contrast topressurized systems, e.g., propellant-driven aerosol sprays, whereactuation is typically achieved by controlled release of pressure e.g.,by controlled opening of a valve.

In certain embodiments the pump sprays are preferred as the use of apump spray with the formulation of the present invention allows for theadministration of droplets or particles having a mean diameter of atleast about 10 microns, preferably at least about 20 microns, morepreferably a mean diameter of from about 20 to about 200 microns, and/orpreferably having a size distribution of from about 5 microns to about500 microns, preferably from about 10 microns to about 200 microns,preferably from about 20 microns to about 100 microns, more preferablyfrom about 30 microns to about 70 microns. This is in contrast to apressurized system which may result in particles less than 5 microns.Liquid droplets or particles having a diameter of less than about 5microns have the potential to enter into the lungs of a human uponadministration. Such entry into the lungs could lead to an increase inpatient to patient variability in absorption of the fentanyl. Further,absorption of fentanyl in the lungs could lead to an increasedabsorption and increased side effects, including respiratory depressionwhich may be fatal.

In certain preferred embodiments, the droplet size of the deliveredformulations further provides for an increase in surface area by beingsprayed sublingually as opposed to being placed under the tongue withe.g., a dropper.

In certain preferred embodiments, the delivery device is a device suchas those described in U.S. Pat. Nos. 6,866,566; 6,877,672; 6,772,915;6,725,857; 6,705,493; 6,679,248; 6,578,741; 6,527,144; 6,484,715;6,478,196; 6,461,322; 6,446,839; 6,427,878; 6,367,473; 6,364,166;6,321,942; 6,234,366; 6,227,413; 6,059,151; 6,059,150; 6,055,979;5,944,222; 5,901,883; 5,813,570; 4,565,302; 4,532,967; 6,964,381;6,860,411; 6,824,020; 6,817,490; 6,585,172; 6,443,370; 6,427,680;6,425,499; 6,401,987; 6,398,074; 6,264,065; 5,950,877; 5,328,099;5,301,846; and the like which are described in certain embodiments asbeing suitable for nasal administration.

Other devices suitable for use in accordance with the formulations ofthe present invention are described in U.S. Pat. Nos. 6,808,085;6,736,293; 6,732,955; 6,708,846; 6,626,379; 6,626,330; 6,626,328;6,454,185; 6,427,876; 6,427,684; 6,419,167; 6,405,903; 6,352,181;6,308,867; 6,257,461; 6,257,454; 6,250,509; 6,227,415; 6,209,760;6,179,164; 6,109,547; 6,062,430; 6,026,992; 5,992,704; 5,992,703;5,988,449; 5,967,369; 5,964,417; 5,950,879; 5,938,125; 5,927,559;5,921,444; 5,893,484; 5,875,938; 5,862,962; 5,860,567; 5,816,504;5,813,570; 5,803,311; 5,791,518, 5,692,650; 5,655,689; 5,584,417;5,520,337; 5,519,980; 5,482,193; 5,469,989; 5,443,185; 5,439,177;5,437,398; 5,427,280; 5,395,032; 5,375,745; 5,368,201; 5,366,122;5,366,122; 5,335,823; 5,326,000; 5,323,936; 5,316,198; 5,301,841;5,295,628; 5,289,946; 5,277,334; 5,257,726; 5,228,586; 5,209,375;5,203,840; 5,147,087; 5,115,980; 5,110,052; 5,011,046; 4,958,752;4,946,069; 4,944,430; 4,934,568; 4,921,142; 4,871,092; 4,830,284;4,826,048; 4,823,991; 4,821,923; 4,817,829; 4,776,498; 4,762,475;4,728,008; 4,726,747; 4,694,977; 4,694,976; 4,566,611; 6,851,583;6,824,021; 6,779,690; 6,776,312; 6,971,559; 6,948,640; 6,945,473;6,938,802; 6,933,850; 6,929,156; 6,918,514; 6,913,205; 6,866,168;6,832,072; 6,830,163; 6,817,490; 6,817,489; 6,811,060; 6,811,057;6,805,301; 6,805,263; 6,789,750; 6,789,706; 6,786,369; 6,783,035;6,772,913; 6,769,579; 6,758,371; 6,752,298; 6,742,677; 6,705,062;6,698,627; 6,698,623; 6,663,019; 6,659,314; 6,659,307; 6,655,550;6,655,549; 6,651,846; 6,601,735; 6,595,395; 6,592,010; 6,588,629;6,581,852; 6,571,991; 6,554,160; 6,536,635; 6,527,149; 6,527,148;6,488,185; 6,471,097; 6,460,781; 6,460,740; 6,460,738; 6,446,841;6,422,429; 6,409,049; 6,398,079; 6,360,919; 6,349,856; 6,345,737;6,343,722; 6,662,561; 6,315,169; 6,273,303; 6,273,300; 6,261,274;6,257,457; 6,234,363; 6,234,168; 6,221,054; 6,209,759; 6,189,741;6,186,371; 6,155,496; 6,119,897; 6,105,826; 6,021,930; 6,012,615;5,988,496; 5,950,871; 5,931,386; 5,850,948; 5,803,318; 5,799,810;5,769,325; RE35,683; 5,692,492; 5,568,884; 5,566,865; 5,511,698;5,482,188; 5,476,198; 5,366,115; 5,337,923; 5,249,713; 5,237,797;5,234,135; 5,226,563; 5,190,192; 5,176,296; 5,127,548; 4,966,313;491,840; 4,245,967; 4,030,667; and the like.

All of the patents recited herein are hereby incorporated by referencein their entireties. Although the delivery devices disclosed in thepatents described above may be suitable for nasal or inhalationadministration, in accordance with certain embodiments of the presentinvention the delivery devices are specifically adapted to be suitablefor sublingual administration of a liquid formulation. In certainembodiments, the devices utilized to practice the present inventioninclude components made from Pfeiffer of America, Inc., for example,Pfeiffer of America, Inc. sublingual unit dose device article referencenumber 72772. In other embodiments, the device is Pfeiffer of America,Inc., sublingual unit dose applicator assembly.

Preferably the device in accordance with the present invention isadapted to sublingually deliver the sublingual formulation in acontrolled manner preferably such that only droplets having a meandiameter of at least about 10 microns, preferably at least about 20microns, more preferably a mean diameter of from about 20 to about 200microns are delivered to the patient. More preferably only dropletshaving a size distribution in the range of from about 5 microns to about500 microns, preferably from about 10 microns to about 200 microns,preferably from about 20 microns to about 100 microns, more preferablyfrom about 30 microns to about 70 microns.

Preferably the dispenser is constructed in such a way that it can becarried and simultaneously reliably operated with the fingers, or withthree fingers of one hand, and can be used, for example, in the mannerof a sublingual spray. The dispenser can be constructed as a disposabledispenser which, following the emptying of the medium chamber, does nothave to be refilled and can therefore be constructed as a simplestandard component, which receives the pump, the formulation, thechannels and optionally, valves or closures within an outer casing,which in side view can be roughly T-shaped or Y-shaped.

If the dispenser is to be emptied in a single pump stroke in successiveportions or in one complete pump stroke, and is not to be refilled, thenthe dispenser can be substantially tightly closed with respect to theoutside in the starting position

In certain preferred embodiments, the delivery device (e.g., such as aspray pump device) includes a lock-out mechanism. Preferably thelock-out mechanism allows for administration of only one unit dose, andpreferably prevents abuse of the fentanyl, a pharmaceutically acceptablesalt thereof, or derivative thereof, by only allowing for theadministration of one dose and locking out of further administration fora certain and/or predetermined period of time. In certain embodiments,after one or more actuating cycles the actuator can be automaticallytransferred into the locking position, so that for performing afollowing actuating cycle randomly or deliberately a release must takeplace. Locking can take place in the starting position, actuatingposition and/or an intermediate position and can act both againstactuation and against return or against one of these movements alone andseveral locking positions with the same or different locking action arepossible.

In certain embodiments, the device may be premetered or alternatively,the device may be device-metered. Premetered devices preferably containpreviously measured doses or a dose fraction in some type of units(e.g., single unit dose amount of solution, single or multiple blistersor other cavities) that may be included in the device during manufactureor by the patient before use. Typical device-metered units have areservoir containing formulation sufficient for multiple doses that aredelivered as metered sprays by the device itself when activated by thepatient.

Important factors to consider with manufacture of the device are thereproducibility of the dose, the spray plume, and the particle/dropletsize distribution, which can affect the delivery of the fentanyl, apharmaceutically acceptable salt thereof, or derivative thereof, underthe tongue. Maintaining the reproducibility of these parameters throughthe expiration dating period and ensuring the functionality of thedevice (e.g., spray mechanism, electronic features, sensors) through itslifetime under patient-use conditions is important as any alteration inthese parameters could lead to variability in dosing and absorption,which could lead to potential side effects.

The administered dose of spray drug formulation may be dependent on thedesign, reproducibility, and performance characteristics of thecontainer closure system. A suitable device which provides the desireddroplet/particle size distribution is an important factor for thecorrect performance of the fentanyl product. Actuation parameters (e.g.,force, speed, hold and return times) should also be considered withrespect to the device. Moreover, the device should be compatible withformulation components. Further, the device should be designed toprevent partial metering of the fentanyl, a pharmaceutically acceptablesalt thereof, or derivative thereof, formulation when used according tothe patient instructions for use.

A typical device includes a base unit, a discharge actuator, an orificefor the formulation to be release from the device, and a mediumreservoir. Preferably a reservoir is provided which as a dispensingchamber is filled already on production of the device. The mediumreservoir preferably defines a measured content of fentanyl, apharmaceutically acceptable salt thereof, or derivative thereof, to bedischarged upon a single activation.

In accordance with certain embodiments of the invention, a reservoir, ora space thereof receiving the medium is preferably an elongated shapepreferably having a wall thickness which is constant over thecircumference and length of the reservoir body. The reservoir body maybe formed simply by a section of a cylindrical hollow of a plastics,steel, such as stainless steel, transparent material, such as glass, orthe like so that its production is very simple.

Preferably an actuator body is provided on a unit of the device, whichis movable relative to the orifice for activating discharge. This body,in the course of the actuating movement, opens a closure of a chamber,e.g. by puncturing. The space within this chamber may directly adjointhe medium in the reservoir, accommodate the opening body or thereservoir at least in part and configured as a pressure space whichprior to being opened is at an elevated pressure. The opening body maybe formed directly by the reservoir.

Preferably during a part of the actuating travel following the startingposition an elevated pressure is built up. In a subsequent portion ofthe actuating movement continuing in the same direction, the medium isrelieved of the pressure at one of the sides and communicated to themedium orifice on this side. As such, due to the pressure acting on theside, the medium is pushed from the reservoir and through the orifice.

Typically as the liquid formulation leaves the orifice, the liquiddroplets follow a trajectory which is influenced by the orifice shape ofthe device. In certain embodiments, the droplet size, spray geometry,and the spray pattern are dependent on the design of the pump and/or theproperties of the formulation. In certain embodiments, the orientationof the actuator, pump design, and the properties of the formulation willinfluence the spray symmetry and the shape.

In certain embodiments, the device of the present invention furtherincludes a stopper. Preferably the stopper comprises a material whichprecludes or substantially precludes the absorption of the fentanyl,pharmaceutically acceptable salt thereof, or derivative thereof. Asuitable stopper for use in accordance with the device of the presentinvention is, for example, a stopper marketed by West PharmaceuticalServices, Inc. In certain preferred embodiments, the stopper has thefollowing composition and characteristic: 1) elastomer: bromobutyland/or chlorobutyl; 2) reinforcement: inert material; and 3) curingsystem: unconventional.

In certain embodiments, the device further includes a gasket. Preferablythe gasket comprises a material which precludes or substantiallyprecludes the absorption of the fentanyl, pharmaceutically acceptablesalt thereof, or derivative thereof. A suitable gasket for use inaccordance with the device of the present invention is, for example, astopper marketed by West Pharmaceutical Services, Inc. In certainpreferred embodiments, the gasket has the following composition andcharacteristic: 1) elastomer: bromobutyl and/or chlorobutyl; 2)reinforcement: inert material; and 3) curing system: unconventional.

Droplet size distribution can be determined by utilizing any reliablemethod known to one of skill in the art. One such method uses laserdiffraction devices, such as, for example, the Malvern® (Malvern is aregistered trademark of Malvern Instruments Limited) Spraytec® with RTSizer Software. A Malvern® Mastersizer® (Mastersizer is a registeredtrademark of Malvern Instruments Limited) S, by Malvern® InstrumentsLimited (U.K.), device may also be used to determine size distribution.A Malvern® Mastersizer® S is a modular particle size analyzer offeringmeasurement versatility. It can measure spray droplet size as well aswet and dry samples. Particles from sub-micron to a few millimeters maybe measured with the Malvern® Mastersizer® S.

Further, automated actuation stations for comparative in vitrobioequivalence tests or other testing to decrease the variabilityassociated with manual actuation may also be used when determining thedroplet size distribution. Any such automated actuation stations knownto one of skill may be applicable in practicing the present invention.An example of one such device is the MightyRunt Actuation Station byInnova Systems, Inc. In a preferred embodiment, a MightyRunt is equippedwith an exhaust fan attachment. In a further embodiment, the MightyRuntis further equipped with a Mettler Toledo® (Mettler Toledo is aregistered trademark of Mettler-Toledo AG) balance Model AT201.

Other Active Agents

Although the invention described herein has been described with respectto fentanyl, a pharmaceutically acceptable salt thereof, or derivativethereof, it is contemplated that other active pharmaceutical agents,particularly those suitable for sublingual administration, may be usedin accordance with the present invention. For example, in certainembodiments, other opioid analgesics which are suitable for sublingualadministration may be used in place of the fentanyl, pharmaceuticallyacceptable salt thereof, or derivative thereof. Certain opioidanalgesics for use in accordance with the present invention include, forexample and without limitation, alfentanil, sufentanil, buprenorphine,butorphanol, codeine, hydrocodone, hydromorphone, levorphanol,meperidine, methadone, morphine, nalbuphine, oxycodone, oxymorphone,propoxyphene, tramadol, and the like.

In certain embodiments the active compound is an analgesic, opioidantagonist, anti-migraine agent, anti-emetic agent, anti-epilepticagent, anti-hypertensive agent, anesthetic agent, cannabinoid,cannabinoid antagonist, inverse agonist of cannabinoid, endocannabinoid,enkephalin, analogues or derivatives thereof, or mixtures thereof.

Analgesics useful in the present invention include, but are not limitedto, alfentanil, buprenorphine, butorphanol, codeine, fenpipramide,hydrocodone, hydromorphone, levorphanol, meperidine, methadone,morphine, nalbuphine, oxycodone, oxymorphone, pentazocine, piritramide,propoxyphene, sufentanil, tilidine, tramadol, analogues or derivativesthereof, or mixtures thereof.

Opioid antagonists useful in the present invention include, but are notlimited to, naloxone, naltrexone, nalmefene, analogues or derivativesthereof, or mixtures thereof.

Anti-migraine agents useful in the present invention include, but arenot limited to, almotriptan, eletriptan, frovatriptan, naratriptan,rizatriptan, sumatriptan, zolmitriptan, ergot alkaloids, proxibarbal,lisuride, methysergide, clonidine, pizotifene, analogues or derivativesthereof, and mixtures thereof.

Anti-emetic agents useful in the present invention include, but are notlimited to bromopride, domperidone, granisetron, ondansetron,tropisetron, metoclopramide, pyridoxine, scopolamine, thiethylperazine,analogues or derivatives thereof, or mixtures thereof.

Anti-epileptic agents useful in the present invention include, but arenot limited to barbiturates, carbamazepine, ethosuximide, mesuximide,phenyloin, primidone, sultiam, valproic acid, vigabatrine, analogues orderivatives thereof, or mixtures thereof.

Anti-hypertensive agents useful in the present invention include, butare not limited to diltiazem, clonidine, nifedipine, verapamil,isosorbide-5-mononitrate, organic nitrates, agents used in treatment ofheart disorders, analogues or derivatives thereof, or mixtures thereof.

Anesthetics useful in the present invention include, but are not limitedto benzocaine, bupivacaine, dibucaine, etidocaine, levobupivacaine,lidocaine, mepivacaine, oxybuprocaine, piperocaine, prilocalne;procaine, proparacaine, ropivacaine, tetracaine, xylocalne, desflurane,enflurane, isoflurane, sevoflurane, benzonatate, dyclonine, ketamine,phenol, propofol, analogues or derivatives thereof, or mixtures thereof.

Cannabinoids useful in the present invention include, but are notlimited to delta-8-tetrahydrocannabinol, delta-9-tetrahydrocannabinol,cannabidol, olivetol, cannabinol, cannabigerol, nabilone,delta-9-tetrahydro cannabinotic acid, the non-psychotropic cannabinoid3-dimethylnepty 11 carboxylic acid homologine 8,delta-8-tetrahydrocannabinol, pharmaceutically acceptable salts thereof,complexes thereof, derivatives thereof, or mixtures thereof. Aparticularly preferred cannabinoid is delta-9-tetrahydrocannabinol, alsoknown as dronabinol.

Further, active agents having a narrow therapeutic index or range (e.g.,wherein small variances in blood levels of the drug causes changes inthe effectiveness or toxicity of that drug) could be particularlysuitable for use in accordance with the present invention. Such activeagents include for example and without limitation, digoxin,levothyroxine, aminoglycosides (e.g., gentamycin, tobramycin),antiarrythimics (e.g., procainamide, quinidine), theophylline,antineoplastics, busulfan, methotrexate, 6-MP, carboplatinum,antidepressants (e.g., lithium), anticonvulsants (e.g., phenyloin,carbamazepine, valproate sodium, valproic acid), antipsychotics,anticoagulants (e.g., warfarin), cyclosporine, and the like.

The present invention will now be more fully described with reference tothe accompanying examples. It should be understood, however, that thefollowing description is illustrative only and should not be taken inany way as a restriction on the generality of the invention specifiedabove.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Example 1

In Example 1, a fentanyl sublingual formulation was prepared having aconcentration of 0.5 mg/ml. The formulation is listed in Table 1 below:

TABLE 1 Ingredient Percent Concentration of Fentanyl percent to make 0.5mg/ml Base Ethanol % (v) 20 Propylene glycol % (v)  5 DI Water % (v) QS

Example 2

In Example 2, a fentanyl sublingual formulation was prepared having aconcentration of 0.5 mg/ml. The formulation is listed in Table 2 below:

TABLE 2 Ingredient Percent Concentration of Fentanyl percent to make 0.5mg/ml Base* Ethanol % (v) 20 Propylene glycol % (v)  5 DI Water % (v) QSContains fentanyl citrate equivalent to 0.5 mg/ml of fentanyl base

Example 3

In Example 3, a fentanyl sublingual formulation was prepared having aconcentration of 0.5 mg/ml. The formulation is listed in Table 3 below:

TABLE 3 Ingredient Percent Concentration of Fentanyl percent to make 0.5mg/ml Base Ethanol % (v) 20 Propylene glycol % (v) 5 DI Water % (v) QSMannitol % (wt) 0.3 Tween 80 % (wt) 0.2

Example 4

In Example 4, a fentanyl sublingual formulation was prepared having aconcentration of 0.5 mg/ml. The formulation is listed in Table 4 below:

TABLE 4 Ingredient Percent Concentration of Fentanyl Base percent tomake 0.5 mg/ml Ethanol % (v) 20 Propylene glycol % (v) 5 Buffer % (v) QSTriacetin % (wt) 0.5

Example 5

In Example 5, a fentanyl sublingual formulation was prepared having aconcentration of 0.5 mg/ml. The formulation is listed in Table 5 below:

TABLE 5 Ingredient Percent Concentration of Fentanyl percent to make 0.5mg/ml Base Ethanol % (v) QS Propylene glycol % (v) 5 Miglyol % (v) 50%

Preparation of Formulations Examples 1-5

-   -   1. Calculated amount of Fentanyl base or Fentanyl citrate was        weighed in a tared glass container.    -   2. Calculated amount of alcohol was added to the container and        mixed to dissolve fentanyl.    -   3. Propylene glycol was weighed and added to the fentanyl        solution.    -   4. Water or Buffer or Miglyol® (Miglyol is a registered        trademark of Cremer Oleo GmbH & Co. KG Limited Liability        Partnership) was weighed, added to the fentanyl solution and        mixed for 2 mm.    -   5. Inactive ingredients (Mannitol, Triacetin, or TW80) were        added at the end and mixed well.    -   6. The final solution was vortexed for 3 min. After mixing, the        formulations were stored in refrigerator for further studies.

Example 6

In Example 6, three rabbits weighing 2-3 kg were used to study thebioavailability of sublingual administration of Examples 1-5 incomparison to the IV injection of the formulation as a reference.Rabbits were first anesthetized by Isoflurane gas as needed to keep therabbits immobilized for approximately 15-20 min.

For each formulation, study rabbits received a single dose of 0.1 ml(equivalent to 50 μg of fentanyl base) by sublingual and IVadministration. For the sublingual studies, the dose of liquidformulation was administered underneath the tongue using a spray bottle.Blood samples (1 ml per sample) were obtained through a catheterinstalled inside the ear vein.

Blood samples were collected at zero time baselines and at 5, 10, 20,45, 60 and 120 min after the single dose. Samples were immediatelycooled and plasma was separated by centrifugation within 2-3 hrs ofblood collection. Samples were stored at −20° C. until assayed. Afterrecovering from anesthesia, animals were returned to their cages. Theseanimals were rested in cages for at least 5-7 days before they could bereused for further testing.

Plasma Collection & Separation

Blood was collected from rabbits in 3 ml tubes containing 7.5% EDTA andcentrifuged at 3,000 RPM for 15 min to remove blood cells and otherinsoluble material. The plasma was decanted into a silanized tube andkept frozen at −20° C. until assay.

Plasma Extraction

For each plasma sample (0.5 ml), 100 μl of Sufentanil (IS) and 100 μL 5MNaOH (for protein denaturation) were added. The Fentanyl was extractedwith 1 ml of 1-chlorobutane by vortex mixing for 15 min. Aftercentrifuging at 12000 RPM for 5 min and freeze-drying for 10 min (tobreak up any emulsion), the upper organic layer was decanted andevaporated to dryness using a gentle stream of nitrogen. The extractionresidue was reconstituted with 100 μl methanol followed by vortex mixingfor 5 min and sonicating for 3 min, then a 3 μl volume was injected intothe GC-MS system.

Results and Discussion

Plasma Concentration Vs. Time Profiles

Plasma concentration-time profiles of Fentanyl in rabbits following IVand sublingual administration of 0.1 ml (equivalent to 50 μg of fentanylbase) are given in the following tables (Tables 6A-10B).

Tables 6A and 6B provide the plasma concentration-time profiles forExample 1.

TABLE 6A (Example 1 intravenous) TIME R1 R2 R3 GRAND (min) (ng/ml)(ng/ml) (ng/ml) AVG STD DEV 5 785.8012 839.9696 812.885396 38.302813 10507.7546 715.6065 611.680527 146.97351 20 737.3449 423.5903 667.9939609.643027 164.81539 45 701.5631 482.215 382.5801 522.119422 163.1925660 562.501 554.8479 475.4686 530.939148 48.191101 90 207.5541 492.4037429.1217 376.359829 149.57498 120 214.0297 196.9675 205.4986 12.064797

TABLE 6B (Example 1 sublingual) TIME R1 R2 R3 GRAND (min) (ng/ml)(ng/ml) (ng/ml) AVG STD DEV 5 412.1095 370.7992 391.454361 29.210825 10205.5274 279.3298 755.3469 413.401352 52.186202 20 539.2677 627.0507712.6511 626.323191 86.693973 45 618.9493 387.7606 503.35497 163.4750660 349.3563 218.14 498.3773 355.291188 140.2129 90 245.8519 249.5091231.7688 242.376606 9.3668848 120 214.1339 162.9939 199.6146 192.24746526.353936

In Tables 7A and 7B are the plasma concentration-time profiles forExample 2.

TABLE 7A (Example 2 intravenous) TIME R1 R2 R3 GRAND (min) (ng/ml)(ng/ml) (ng/ml) AVG STD DEV 5 671.5152 788.2252 729.870183 58.35497 10379.1866 617.5517 1102 699.579446 300.73378 20 603.9696 407.5375505.75355 98.216024 45 380.9878 292.998 336.992901 43.994929 60 140.7566266.3611 388.5314 265.216362 101.1569 90 81.06491 254.4848 334.8012223.450304 105.88636 120 95.34888 278.7789 232.4037 202.177147 77.875432

TABLE 7B (Example 2 sublingual) TIME R1 R2 R3 GRAND (min) (ng/ml)(ng/ml) (ng/ml) AVG STD DEV 5 350.2297 106.9249 337.6856 264.946755136.99455 10 373.5822 115.1643 486.0254 324.923935 190.15835 20 285.5994294.5517 518.5091 366.220081 131.96212 45 302.7099 52.78093 359.9483238.479716 163.34652 60 118.3915 43.02434 314.9564 158.790737 140.3952890 81.06491 43.39148 62.2281947 26.639136 120 95.34888 35.34888 30.6815453.7931034 36.063946

In Tables 8A and 8B are the plasma concentration-time profiles forExample 3.

TABLE 8A (Example 3 intravenous) TIME (min) R1 (ng/ml) R2 (ng/ml) R3(ng/ml) GRAND AVG STD DEV 5 903.4949 1000.205 882.0649 928.58823551.390778 10 814.3834 557.9432 484.5233 618.949966 141.40552 20 348.8641309.1785 329.021298 19.842799 45 120.2677 169.7972 211.0669 167.04394937.119701 60 160.3347 121.5882 128.9087 136.943881 16.807631 90 89.8559885.70081 92.71197 89.4229209 2.8786265 120

TABLE 8B (Example 3 sublingual) TIME R1 R2 R3 GRAND (min) (ng/ml)(ng/ml) (ng/ml) AVG STD DEV 5 127.3124 269.1927 198.252535 100.32454 10304.6288 589.1684 310.5953 401.464165 162.58397 20 349.3611 689.7999281.5365 440.232477 218.77603 45 288.0639 418.1555 195.9391 300.719518111.64744 60 173.0345 255.9016 214.45 74.967507 90 224.432 87.7931228.6156 180.280257 80.123534 120 96.5284 145.1907 120.859533 34.409422

In Tables 9A and 9B are the plasma concentration-time profiles forExample 4.

TABLE 9A Example 4 intravenous) TIME R2 GRAND (min) R1(ng/ml) (ng/ml) R3(ng/ml) AVG STD DEV 5 740.3759 732.5855 717.8438 730.268425 11.443374 10666.2644 671.4571 627.0568 654.926076 24.274753 20 713.355 592.5638.7444 648.199797 60.979784 45 575.2667 557.7789 482.8316 538.62576149.103907 60 409.8756 596.6278 548.1217 518.208362 96.903054 90 455.8567484.928 430.5538 457.112801 27.208875 120 385.2982 452.6998 418.99898647.660144

TABLE 9B (Example 4 sublingual) TIME R1 R2 R3 GRAND (min) (ng/ml)(ng/ml) (ng/ml) AVG STD DEV 5 250.8854 481.6146 459.2779 397.259297127.25455 10 478.074 577.9594 614.8479 556.960446 70.76361 20 587.8195465.9229 518.4341 524.058824 61.142626 45 538.3245 499.217 449.574495.705206 44.479353 60 367.4361 452.9817 442.7566 421.058147 46.71863690 309.5538 571.0487 202.8722 361.158215 189.43533 120 205.6531 195.7282427.1927 276.191346 130.86513

In Tables 10A and 10B are the plasma concentration-time profiles forExample 5.

TABLE 10A (Example 5 intravenous) TIME (min) R1 (ng/ml) R2 (ng/ml) R3(ng/ml) GRAND AVG STD DEV 5 928.5193 1247.247 1123.335 1099.70047160.67311 10 920.1521 1100.3 1103.844 1041.43205 105.04643 20 876.3793966.998 972.8114 938.729547 54.075067 45 765.9696 938.3834 947.0609883.804598 102.14032 60 645.6045 892.6836 482.2799 673.52265 206.6212990 157.9533 418.6034 343.0811 306.545977 134.1109 120 58.43813 30.6815430.68154 39.933739 16.025276

TABLE 10B (Example 5 sublingual) TIME R1 R2 R3 GRAND (min) (ng/ml)(ng/ml) (ng/ml) AVG STD DEV 5 140.7911 176.7338 127.357 148.29394925.529127 10 239.712 191.2956 210.8458 213.951149 24.357082 20 409.5335580.6095 540.5842 510.242394 89.483087 45 351.3955 500.9315 314.7343389.020453 98.636103 60 364.9493 439.2885 280.645 361.62762 79.373899 90245.2698 403.4255 30.68154 226.458925 187.08258 120 30.68154 119.7079215.6552 122.014875 92.508392Pharmacokinetic Parameters

The pharmacokinetic parameters Peak plasma concentration (Cmax), Time toreach Cmax(Tmax), Half-life (t_(1/2)), Area under the curve (AUC), andTotal body clearance (CL) obtained after sublingual or IV drugadministrations are tabulated in the following tables (Tables 11-15).Also, plasma concentration-time curves after administering IV andsublingual doses of Fentanyl are shown in FIGS. 1-10.

TABLE 11 (Example 1 intravenous and sublingual) ROUTE OF AUC T_(1/2)TMAX CMAX VD Cl ADMINISTRATION (min*ng/ml) (min) (min) (ng/ml) (ml)(ml/min) IV 71772.86 43.8141 0 812.8854 50.7843 0.6966 SL 59684.3455.704 20 626.3232

TABLE 12 (Example 2 intravenous and sublingual) ROUTE OF AUC T_(1/2)TMAX CMAX VD Cl ADMINISTRATION (min*ng/ml) (min) (min) (ng/ml) (ml)(ml/min) IV 86790.18 153.2372 0 729.8702 181.8356 0.9218 SL 23759.1433.1477 20 366.2201

TABLE 13 (Example 3 intravenous and sublingual) ROUTE OF AUC T_(1/2)TMAX CMAX VD Cl ADMINISTRATION (min*ng/ml) (min) (min) (ng/ml) (ml)(ml/min) IV 32707.76 49.7523 0 928.5882 78.4776 1.5287 SL 40623.1357.6038 20 440.2325

TABLE 14 (Example 4 intravenous and sublingual) ROUTE OF AUC T_(1/2)TMAX CMAX VD Cl ADMINISTRATION (min * ng/ml) (min) (min) (ng/ml) (ml)(ml/min) IV 185485.6 200.3556 0 730.2684 77.036 0.2696 SL 86916.9193.4018 10 556.9604

TABLE 15 (Example 5 intravenous and sublingual) ROUTE OF AUC T_(1/2)TMAX CMAX VD Cl ADMINISTRATION (min * ng/ml) (min) (min) (ng/ml) (ml)(ml/min) IV 76250.66 17.0944 0 1099.7005 27.3544 0.6557 SL 42554.5938.2788 20 510.2424

The maximum concentrations were reached in about 20 minutes for allformulations after sublingual administration. There was considerableinter-individual variability by both routes of administration.Measurable plasma concentration after sublingual administration wasdemonstrable after 120 min in most formulation testing. In conclusion,sublingual fentanyl administration showed good absorption profilecompared to IV.

Example 7

In Example 7, a study was conducted to determine the pharmacokinetics ofa formulation prepared in accordance with Example 1 after increasingsublingual dose administration in healthy volunteers under fastingconditions. The study was also conducted to determine the safety andtolerability of a fentanyl sublingual spray prepared in accordance withExample 1 in humans.

The study was a single center, single dose, single-blinded, sequentialascending dose and repeated design in healthy male subjects. Thefollowing treatments were to be administered under fasting conditions:

Treatment A (Test 1): One Fentanyl 1 mg/ml sublingual spray (1×100 μgdose)

Treatment B (Test 2): One Fentanyl 4 mg/ml sublingual spray (1×400 μgdose)

Treatment C (Test 2): Two Fentanyl 4 mg/ml sublingual spray (1×800 μgdose)

Treatment D (Placebo-Test 1): One Placebo 1 mg/ml sublingual spray(1×100 μg dose)

Treatment E (Placebo-Test 2): One Placebo 4 mg/ml sublingual spray(1×400 μg dose)

Treatment F (Placebo-Test 2): Two Placebo 4 mg/ml sublingual spray(1×800 μg dose)

The products were to be administered to nine (9) healthy male volunteersaccording to the following design in Table 16 below:

TABLE 16 Period 1 Period 2 Period 3 Sequence 1 (n = 6) 100 μg 400 μg 800μg Sequence 2 (n = 3) Placebo Placebo Placebo

In each period, subjects were to arrive at the clinical site at least 10hours before dosing. After a supervised overnight fast, a single oraldose of the assigned formulation was to be orally administered in themorning. Subjects were allowed to leave the clinical site after the24-hour blood draw. The wash-out period was to be of at least 14 days;the duration of this study was expected to be approximately 5-6 weeks.As per protocol, each sequential dose was to be separated by a wash-outof at least 14 days, which corresponds to more than 10 time the expectedhalf life of the moiety to be measured. However, during the studyperiods 1 and 2 were separated by a wash-out of 15 days and periods of 2and 3 were separated by a wash-out of 13 days. As fentanyl's expectedhalf life is reported to be approximately 6.4 hours, it is judged thatthese wash-out deviations should not affect the conclusion of the study.Furthermore, no carry-over was observed at the beginning of the secondand third periods.

Pharmacokinetic Assessments

Blood samples for pharmacokinetic measurements were collected prior toand up to 24 hours (serial samplings) after each drug administration.The drug concentrations produced by the administration of the studiedformulations were used to derive the pharmacokinetic parameters listedin Tables 17 and 18 below.

Six (6) subjects were included in the statistical analysis. A summary ofthe non-normalized pharmacokinetic parameters is presented in Table 17and a summary of the normalized pharmacokinetic parameters is presentedin Table 18. The mean measured plasma concentrations versus timeprofile, produced by the administration of the Test products, isdepicted in FIG. 11, whereas the ln-transformed mean concentrationsversus time profile is presented in FIG. 12.

The pharmacokinetic parameters of interest for this study were C_(max),AUC_(∞), AUC_(T), AUC_(T/∞), K_(el), T_(max), T_(1/2el), Cl/F andV_(Z)/F.

TABLE 17 Pharmacokinetic Parameters Fentanyl (n = 6) Non-normalized DataTEST 1 (100 μg) TEST 2 (400 μg) TEST 2 (800 μg) n = 6 n = 6 N = 2PARAMETER MEAN C.V. (%) MEAN C.V. (%) MEAN C.V. (%) C_(max) (pg/ml)172.0 27.1 708.0 50.2 1270.4 37.7 ln (C_(max)) (pg/ml) 5.1207 4.8 6.45098.2 7.1102 5.4 T_(max) (hours) 0.50 29.7 0.50 61.3 0.75 0.0 AUC_(T) (pg· h/ml) 472.6 66.2 3556.1 63.0 5417.3 30.6 ln (AUC_(T)) (pg · h/ml)6.0271 8.6 8.0208 7.5 8.5734 3.6 AUC_(∞) (pg · h/ml) 817.9 36.1 4242.657.6 5726.8 28.8 ln (AUC_(∞)) (pg · h/ml) 6.6607 4.8 8.2303 6.4 8.63173.4 AUC_(T/∞) (%) 54.90 28.7 81.48 10.6 94.35 1.8 K_(el) (hour⁻¹) 0.200827.4 0.1593 44.9 0.1782 0.9 T_(1/2 el) (hours) 3.70 30.4 5.20 45.8 3.890.9 Cl/F (ml/h/kg) 1718.8 27.7 1532.2 49.7 1837.3 13.1 V_(z)/F (ml/kg)9070.2 34.7 10470.4 47.1 10307.7 12.2 For T_(max), the median ispresented and the statistical analysis is based on ranks.

TABLE 18 Data Normalized to the 100 μg dose TEST 1 (100 μg) TEST 2 (400μg) TEST 2 (800 μg) n = 6 n = 6 N = 2 PARAMETER MEAN C.V. (%) MEAN C.V.(%) MEAN C.V. (%) C_(max) (pg/ml) 172.0 27.1 177.0  50.2 158.8  37.7 ln(C_(max)) (pg/ml) 5.1207 4.8   5.0646 10.4   5.0307 7.7 T_(max) (hours)0.50 29.7  0.50 61.3  0.75 0.0 AUC_(T) (pg · h/ml) 472.6 66.2  889.0¹ 63.0 677.2  30.6 ln (AUC_(T)) (pg · h/ml) 6.0271 8.6    6.6346¹ 9.1  6.4940 4.8 AUC_(∞) (pg · h/ml) 817.9 36.1 1060.7   57.6 715.9  28.8 ln(AUC_(∞)) (pg · h/ml) 6.6607 4.8   6.8440 7.7   6.5523 4.5 AUC_(T/∞) (%)54.90 28.7 81.48² 10.6  94.35² 1.8 K_(el) (hour⁻¹) 0.2008 27.4   0.159344.9   0.1782 0.9 T_(1/2el) (hours) 3.70 30.4  5.20 45.8  3.89 0.9 Cl/F(ml/h/kg) 1718.8 27.7 1532.2   49.7 1837.3   13.1 V_(Z)/F (ml/kg) 9070.234.7 10470.4   47.1 10307.7   12.2 For T_(max), the median is presentedand the statistical analysis is based on ranks. ¹Different than Test - 1(p < 0.05) ²Different than Test - 1 (p < 0.01) N.S. = Not Significant(p > 0.05) Definition of the Pharmacokinetic Parameters C_(max): Maximumplasma concentration (ng/ml). T_(max): Time of maximum measured plasmaconcentration; if it occurs at more than one time point, T_(max) isdefined as the first time point with this value (hour). C_(LQC): Lastquantifiable concentration (ng/ml). T_(LQC): Time of last quantifiableplasma concentration (hour). AUC_(T): Cumulative Area Under the plasmaconcentration time Curve calculated from 0 to T_(LQC) using theTrapezoidal method (ng · h/ml), and calculated according the followingequation:${AUC}_{T} = {\sum\limits_{2}^{N_{TLQC}}\lbrack {( \frac{C_{n - 1} + C_{n}}{2} ) \times ( {T_{n} - T_{n - 1}} )} \rbrack}$Where N_(TLQC) is the number of the sample related to the T_(LQC).AUC_(∞): Extrapolated Area Under the plasma concentration time Curve toinfinity (ng · h/ml).${AUC}_{\infty} = {{AUC}_{T} + ( \frac{C_{LQC}}{K_{el}} )}$AUC_(T/∞): Relative percentage of AUC_(T) with respect to AUC_(∞) (%).${AUC}_{T/\infty} = {( \frac{{AUC}_{T}}{{AUC}_{\infty}} ) \times 100}$T_(LIN): Time point where log-linear elimination begins (hour). K_(el):Apparent mean elimination rate constant estimated by a non-linearleast-squares regression analysis; a minimum of three values arerequired at the end of the concentration-time curve (hour⁻¹). T_(1/2el):Half-life of elimination (hour). $T_{{1/2}{el}} = \frac{\ln(2)}{K_{el}}$Cl/F: Apparent clearance (ml/h/kg)${{Cl}\text{/}F} = \frac{{Dose}^{*}}{{AUC}_{\infty}}$ V_(Z)/F: Apparentvolume of distribution (ml/kg)${V_{Z}\text{/}F} = \frac{{Dose}^{*}}{K_{el}\mspace{11mu}\bullet\mspace{11mu}{AUC}_{\infty}}$*Dose expressed per weight (kg)

As noted in Table 11, the parameters for which a statisticallysignificant difference was observed between the 100 μg dose and thenormalized 400 μg dose was AUC_(T), ln(AUC_(T)) and AUC_(T/∞) and theparameter for which a statistically significant difference was observedbetween the 100 μg dose and the normalized 800 μg dose was AUC_(T/∞). Nostatistically significant differences were observed for any of the otherpharmacokinetic parameters under study.

The mean C_(max) value of the 100 μg dose was 172.0 pg/ml while for the400 μg dose, it was 708.0 pg/ml and for the 800 μg dose, it was 1270.4pg/ml. Once normalized to the 100 μg dose, the mean C_(max) value of the400 μg dose was 177.0 pg/ml and for the 800 μg dose, it was 158.8 pg/ml.

The median T_(max) was 0.50 hour for the 100 μg dose and the 400 μgdose, and 0.75 hour for the 800 μg dose.

The mean AUC_(T) value of the 100 μg dose was 472.6 pg·h/ml while forthe 400 μg dose, it was 3556.1 pg·h/ml and for the 800 μg dose, it was5417.3 pg·h/ml. Once normalized to the 100 μg dose, the mean AUC_(T)value of the 400 μg dose was 889.0 pg·h/ml and for the 800 μg dose, itwas 677.2 pg·h/ml.

The mean AUC_(∞) value of the 100 μg dose was 817.9 pg·h/ml while forthe 400 μg dose, it was 4242.6 pg·h/ml and for the 800 μg dose, it was5726.8 pg·h/ml. Once normalized to the 100 μg dose, the mean AUC_(∞)value of the 400 μg dose was 1060.7 pg·h/ml and for the 800 μg dose, itwas 715.9 pg·h/ml.

The AUC_(T/∞) ratio was approximately 55% for the 100 μg dose, 81% forthe 400 μg dose and 94% for the 800 μg dose.

The mean K_(el) was 0.2008 hour⁻¹ for the 100 μg dose, 0.1593 hour⁻¹ forthe 400 μg dose and 0.1782 hour⁻¹ for the 800 μg dose, while the meanT_(1/2el) value was 3.70 hours, 5.20 hours and 3.89 hours following thesame order.

The mean Cl/F was 1718.8 ml/h/kg for the 100 μg dose, 1532.2 ml/h/kg forthe 400 μg dose and 1837.3 ml/h/kg for the 800 μg dose, while the meanV_(Z)/F was 9070.2 ml/kg for the 100 μg dose, was 10470.4 ml/kg for the400 μg dose and was 10307.7 ml/kg for the 800 μg dose.

The intra-subject variation was 36.79%, 37.05% and 28.88% for C_(max),AUC_(T), and AUC_(∞), respectively.

Safety Evaluation

The safety parameters included the occurrence of adverse effects,measurements of vital signs, respiratory rate, oxygen saturation ofblood by finger pulse oximetry, ECG and clinical laboratory parameters.

All adverse events were spontaneously reported by the volunteer,observed by the Clinical Investigator (or delegates) or elicited bygeneral questioning by the clinical staff. Adverse events were alsoreported upon completion of the form “Taste of Medication Questionnaireto Subject”, which was filled right after dosing. For the purposes ofthe study, the period of observation for each individual subjectextended from the time the subject gave informed consent to within 7days following the last drug administration.

Safety Results:

All nine subjects experienced a total of one-hundred-twenty-seven (127)adverse events during the study. No serious adverse events were recordedin this study. Twenty adverse events (8 different types) were reportedafter the single dose administration of the Test 1 (A) product,fifty-six adverse events (26 different types) were reported after thesingle dose administration of the Test 2 (B) product, twenty-two adverseevents (19 different types) were reported after the single doseadministration of the Test 2 (C) product, ten adverse events (8different types) were reported after the single dose administration ofthe Placebo-Test 1 (D) product, eleven adverse events (9 differenttypes) were reported after the single dose administration of thePlacebo-Test 2 (E) product and twelve adverse events (8 different types)were reported after the single dose administration of the Placebo-Test 2(F) product. Two (2) adverse events associated with post-studylaboratory test results were imputed to three formulations.

Six subjects (100%) reported adverse events after the administration ofthe Test 1 (A) formulation, six subjects (100%) reported adverse eventsafter the administration of the Test 2 (13) formulation, two subjects(100%) reported adverse events after the administration of the Test 2(C) formulation, three subjects (100%) reported adverse events after theadministration of the Placebo-Test 1 (D) formulation, three subjects(100%) reported adverse events after the administration of thePlacebo-Test 2 (E) formulation and three subjects (100%) reportedadverse events after the administration of the Placebo-Test 2 (F)formulation. The adverse events by system Organ Class are listed inTable 19.

The events abdominal distension, abdominal pain, abdominal pain upper,anxiety, depressed mood, diarrhea, disturbance in attention, dizziness(10 episodes out of 11), dry mouth, dry skin, dysgeusia, headache,fatigue (6 episodes out of 7), feeling cold, feeling drunk, feeling hot,feeling of relaxation, hot flush, hyperhidrosis, hypoaesthesia oral,hypoaesthesia, nasal congestion, nausea, oral discomfort, pallor,paresthesia oral, pruritus, sensation of heaviness, somnolence, speechdisorder, tongue coated and vomiting were assessed to be possiblyrelated to the drugs. The other events cough, dizziness (1 episode outof 11), fatigue (1 episode out of 7), musculoskeletal pain, rhinorrhoeaand throat irritation were assessed to be not related to the studydrugs. The other event nasopharyngitis was assessed to be unlikelyrelated to the study drugs.

TABLE 19 Summary of Adverse Events by System Organ Class Period 1 Period2 Period 3 Fentanyl Fentanyl Fentanyl Parameter 100 μg Placebo 400 μgPlacebo 800 μg Placebo Number of subjects exposed 6 3 6 3 2 3 Number ofsubjects reporting at 6 3 6 3 2 3 least one adverse event Total numberof withdrawals 0 0 4 0 0 0 Withdrawals due to adverse event 0 0 1 0 0 0(not related to tested drug) Withdrawals due to adverse event 0 0 2 0 00 (related to tested drug) Total number of adverse events* 20 10 56 1122 12 Adverse events at least possibly 18 8 53 11 22 12 drug related*Number % of Number % of Number % of Number % of Number % of Number % ofof subjects of subjects of subjects of subjects of subjects of subjectsNature of adverse events subjects exposed subjects exposed subjectsexposed subjects exposed subjects exposed subjects exposedGastrointestinal disorders Abdominal distension 0 0 0 0 1 17 0 0 0 0 0 0Abdominal pain 0 0 0 0 0 0 1 33 0 0 1 33 Abdominal pain upper 0 0 0 0 117 0 0 0 0 0 0 Diarrhoea 0 0 0 0 0 0 1 33 0 0 1 33 Dry mouth 0 0 0 0 0 00 0 1 50 0 0 Dysgeusia 6 100 3 100 6 100 3 100 2 100 3 100 Hypoaesthesiaoral 4 67 1 33 3 50 1 33 1 50 0 0 Nausea 0 0 0 0 4 67 0 0 1 50 0 0 Oraldiscomfort 1 17 0 0 1 17 1 33 1 50 0 0 Tongue coated 0 0 0 0 1 17 0 0 00 0 0 Vomiting 0 0 0 0 1 17 0 0 1 50 0 0 General disorders andadministration site conditions Fatigue 3 50 1 33 2 33 0 0 1 50 0 0Feeling cold 0 0 0 0 1 17 0 0 1 50 0 0 Feeling drunk 0 0 0 0 6 100 0 0 00 0 0 Feeling hot 0 0 0 0 1 17 0 0 1 50 0 0 Feeling of relaxation 3 50 00 1 17 0 0 0 0 0 0 Hyperhidrosis 0 0 0 0 0 0 0 0 1 50 0 0Investigations* Alanine aminotransferase increased 0 0 1 33 0 0 1 33 0 01 33 Aspartate aminotransferase 0 0 1 33 0 0 1 33 0 0 1 33 increasedMusculoskeletal and connective tissue disorders Sensation of heaviness 00 0 0 1 17 0 0 0 0 0 0 Musculoskeletal pain 0 0 0 0 1 17 0 0 0 0 0 0Nervous system disorders Dizziness 0 0 0 0 6 100 0 0 2 100 0 0Disturbance in attention 0 0 0 0 2 33 0 0 1 50 0 0 Headache 0 0 0 0 1 170 0 0 0 1 33 Hypoaesthesia 0 0 0 0 4 67 0 0 1 50 0 0 Paresthesia oral 00 1 33 3 50 1 33 0 0 3 100 Somnolence 0 0 1 33 1 17 0 0 2 100 1 33Speech disorder 0 0 0 0 0 0 0 0 1 50 0 0 Psychiatric disorders Anxiety 00 0 0 0 0 1 33 0 0 0 0 Depressed mood 0 0 0 0 1 17 0 0 0 0 0 0Respiratory, thoracic and mediastinal disorders Cough 1 17 0 0 0 0 0 0 00 0 0 Nasal congestion 1 17 0 0 1 17 0 0 0 0 0 0 Nasopharyngitis 0 0 133 0 0 0 0 0 0 0 0 Rhinorrhoea 1 17 0 0 0 0 0 0 0 0 0 0 Throatirritation 0 0 0 0 1 17 0 0 0 0 0 0 Skin and subcutaneous tissuedisorders Dry skin 0 0 0 0 0 0 0 0 1 50 0 0 Pallor 0 0 0 0 1 17 0 0 1 500 0 Pruritus 0 0 0 0 0 0 0 0 1 50 0 0 Vascular disorders Hot flush 0 0 00 3 50 0 0 1 50 0 0 *= Two (2) adverse events were associated withpost-study laboratory test results and have been assigned to eachperiod.Conclusions

The pharmacokinetic parameters were well defined for the three doses(100 μg, 400 μg and 800 μg) administered in this study. C_(max) andAUC_(∞) seem to be proportional, AUC_(T) is consistent withdose-proportionality between the 400 μg and 800 μg. Furthermore, the twoformulations of fentanyl (1 mg/ml and 4 mg/ml sublingual spray in dosesof 100 μg, 400 μg and 800 μg) administered during the study were welltolerated in most of the subjects. No subject participating in the trialreported serious adverse events during the course of this study.

Examples 8-12 In-Vitro Permeation Experiments

The permeation characteristics of fentanyl formulations were studiedusing EpiOral tissues (ORL-100). MatTek Corp's EpiOral is used as modelfor screening sublingual drug absorption of pharmaceutical formulations.MatTek's EpiOral tissue consists of normal, human-derived epithelialcells. The cells have been cultured to form multilayered, highlydifferentiated models of the human buccal (EpiOral) phenotypes. TheEpiOral tissue model exhibits in vivo-like morphological and growthcharacteristics which are uniform and highly reproducible.Morphologically, the tissue model closely parallels native human tissue,thus providing a useful in-vitro means to assess in-vivo permeability ofpharmaceutical formulations across sublingual mucosa.

The EpiOral tissues, grown on cell culture inserts with Teflon® (Teflonis a registered trademark of E.I. Du Pont De Nemours and Company)backing membrane, were shipped by MatTek Corp on Monday for delivery onTuesday morning. All the tissues were used in the permeabilityexperiments within 72 hours of shipment. The inserts containing thetissues were rinsed with distilled water before the start of permeationexperiments. The tissue area for the ORL-100 is 0.6 cm2.

The receiver compartment (wells) received 0.3 ml of phosphate citratebuffer of pH 6.6 (receiver solution). The donor compartments (tissueinserts) were placed in the wells and filled with 0.5 ml of drugsolution (donor solution).

The inserts were moved from well to well containing fresh receiver fluidat predetermined intervals (2, 5, 7, 9, 11, 13, 15, 30, 45, 60, 90, and120 min). After permeation studies both donor and receiver fluids werecollected in vials for analysis by HPLC. The receiver and donor solutionconcentrations and the flux over each permeation time interval weredetermined.

Example 8

In Example 8, the permeation of fentanyl base was compared to thepermeation of fentanyl citrate. The formulations and percent permeatedin 2 hours is listed in the table below:

TABLE 20 CONC. OF % FENTANYL ALCOHOL PG MIGLYOL ® PERMEATED EXAMPLE # *BASE % (V) % (V) % (V) IN 2 HOURS Fentanyl 8-a(b) 1 mg/ml 20 5 — 17.33Base Fentanyl 8-b(w) 1 mg/ml 20 5 — 17.18 Base Fentanyl 8-c(b) 0.646mg/ml    20 5 — 1.81 Citrate Fentanyl 8-d 1 mg/ml 20 5 79.3 1.64 Base(b)—buffer, (w)—water

Example 9

In Example 9, Effect of Alcohol on the Permeation of Fentanyl BaseFormulations was tested. The formulations and percent permeated in 2hours are listed in the Table below:

TABLE 21 CONC. OF % FENTANYL ALCOHOL PG PERMEATED IN EXAMPLE #* BASE %(V) % (V) 2 HOURS 9-a (w) 1 mg/ml 20 5 17.18 (Control) 9-b (w) 1 mg/ml28 5 13.45 9-c (w) 1 mg/ml 40 5 10.95 9-c (w) 1 mg/ml 50 5 9.18(b)—buffer, (w)—water

Example 10

In Example 10, the effects of PG (propylene glycol) on FentanylFormulations was tested. The formulations and percent permeated in 2hours are listed in the table below:

TABLE 22 CONC. OF % FENTANYL ALCOHOL PG PERMEATED IN EXAMPLE #* BASE %(V) % (V) 2 HOURS 10-a (w) 1 mg/ml 20 5 17.18 (Control) 10-b (w) 1 mg/ml20 25 14.518 (b)—buffer, (w)—water

Example 11

In Example 11, the effect of pH on Fentanyl Formulations was tested. Theformulations and percent permeated in 2 hours are listed in the tablebelow. As indicated in the table, the permeation of fentanyl acrossbuccal tissue was dependent on the pH of the formulation. Becausefentanyl has pKa value (7.3 and 8.4) within the pH range studied, itsdegree of ionization changed as the pH of the formulation was altered.The results in the table below indicate that that formulations adjustedto pH between 8 and 9 were showing better permeability and physicalstability.

TABLE 23 CONC. OF ALCO- % EXAM- FENTANYL BUFFER HOL PG PERMEATED PLE #*BASE PH % (V) % (V) IN 2 HOURS 11-a (w) 2 mg/ml Water 30 5 17.83(Control) 11-b (b) 2 mg/ml 5.5 30 4 5.33 11-c (b) 2 mg/ml 6.6 29 4 10.6211-d (b) 2 mg/ml 8.6 29 4 13.48 11-e (b) 2 mg/ml 9.6 29 4 11.8(b)—buffer, (w)—water

Example 12

In Example 12, several ingredients including hydroxypropyl betacyclodextrin (HPBCD), mannitol, polyvinyl pyrrolidone (PVP), propylenecarbonate (PC), sodium glycocholate (SG), sodium lauryl sulphate (SLS),triacetin, triethyl citrate and Tween® 80 (Tween is a registeredtrademark of Uniqema Americas LLC; TW 80) were added to the formulationseither individually or in combination and studied for their effect onpermeability and solution stability. Table 24 to 36 summarizes theformulations and permeation results of buffered and water formulationscontaining the above excipients.

TABLE 24 Results of the effects of Buffer and Water on FentanylFormulations. CONC. OF INACTIVE INGREDIENTS % EXAMPLE FENTANYL BUFFERALCOHOL PG % Mannitol Triacetin TW80 PERMEATED #* BASE OR WATER % (V)(V) % (wt) % (wt) % (wt) IN 2 HOUR 12-a(b) 1 mg/ml B 20 5 — — — 17.33(Control) 12-b(w) 1 mg/ml W 20 5 — — — 17.18 (Control) 12-c(b) 1 mg/ml B20 5 0.1 0.15 — 16.14 12-d(w) 1 mg/ml W 20 5 0.1 0.15 — 12.10 12-e(b) 1mg/ml B 20 5 0.3 — 0.2 16.02 12-f(w) 1 mg/ml W 20 5 0.3 — 0.2 12.3512-g(b) 1 mg/ml B 20 5 0.15 0.3  0.1 16.43 12-h(w) 1 mg/ml W 20 5 0.150.3  0.1 9.76 12-i(b) 1 mg/ml B 20 5 0.2 — 0.2 18.74 12-j(w) W 20 5 0.2— 0.2 13.19 12-k(b) 1 mg/ml B 20 5 — — 0.3 14.73 12-l(w) 1 mg/ml W 20 5— — 0.3 13.76 (b)—buffer, (w)—water

The results in Table 24 indicate that all the buffered formulations hadsimilar permeability characteristics as that of control formulationsexcept the buffered formulation containing 0.3% Tween® 80 which showedlower permeability. All water formulations exhibited lower permeabilitythan buffered formulations.

TABLE 25 Effect of HPBCD on Fentanyl Formulations HPBCD CONC. OF(MOLECULAR % FENTANYL ALCOHOL PG RATIO TO PERMEATED EXAMPLE #* BASE %(V) % (V) API) IN 2 HOURS 12-m (w) 1 mg/ml 20 5 — 17.18 (Control) 12-n(w) 1 mg/ml 20 5 1:1 (0.413%) 14.28 12-o (w) 1 mg/ml 20 5 1:2 (0.826%)13.50 (b)—buffer, (w)—water

TABLE 26 Effect of Mannitol on Fentanyl Formulations CONC. OF ALCO- PG %EXAMPLE FENTANYL HOL % MANNITOL PERMEATED #* BASE % (V) (V) % (WT) IN 2HOURS 12-p (b) 1 mg/ml 20 5 — 17.33 (Control) 12-q (b) 1 mg/ml 20 5 0.317.71 12-r (b) 1 mg/ml 20 5 0.4 16.86 12-s (b) 1 mg/ml 20 5 0.5 15.4112-t (b) 1 mg/ml 20 5 0.8 14.81 (b)—buffer, (w)—water

TABLE 27 Effect of Polyvinyl Pyrrolidone (PVP) on Fentanyl FormulationsCONC. OF AL- % EXAM- FENTANYL COHOL PG PVP PERMEATED PLE #* BASE % (V) %(V) % (WT) IN 2 HOURS 12-u (w) 1 mg/ml 20 5 — 17.18 (Control) 12-v (w) 1mg/ml 20 5 0.1 16.73 12-w (w) 1 mg/ml 20 5 0.5 14.68 12-x (w) 1 mg/ml 205 1 14.52 12-y (w) 1 mg/ml 25 5 3 10.75 (b)—buffer, (w)—water

TABLE 28 Effect of Propylene Carbonate (PC) on Fentanyl FormulationsCONC. OF ALCO- % EXAMPLE FENTANYL HOL PG PC PERMEATED #* BASE % (V) %(V) % (WT) IN 2 HOURS 12-z (w) 1 mg/ml 20 5 — 17.18 (Control) 12-aa (w)1 mg/ml 20 5 1 14.39 12-bb (w) 1 mg/ml 20 5 1.5 14.43 (b)—buffer,(w)—water

TABLE 29 Effect of Sodium Glycocholate (SG) on Fentanyl FormulationsCONC. OF ALCO- % EXAMPLE FENTANYL HOL PG SG PERMEATED #* BASE % (V) %(V) % (WT) IN 2 HOURS 12-cc (w) 1 mg/ml 20 5 — 17.18 (Control) 12-dd (w)1 mg/ml 20 5 0.5 18.30 12-ee (w) 1 mg/ml 20 5 1 19.78 (b)—buffer,(w)—water

TABLE 30 Effect of Triacetin on Fentanyl Formulations CONC. OF ALCO- PG% EXAMPLE FENTANYL HOL % TRIACETIN PERMEATED #* BASE % (V) (V) % (WT) IN2 HOURS 12-ff (b) 1 mg/ml 20 5 — 17.33 (Control) 12-gg (b) 1 mg/ml 20 50.5 17.71 12-hh (b) 1 mg/ml 20 5 2 14.56 (b)—buffer, (w)—water

TABLE 31 Effect of Triethyl Citrate on Fentanyl Formulations CONC. OFALCO- PG TRIETHYL % EXAMPLE FENTANYL HOL % CITRATE PERMEATED #* BASE %(V) (V) % (WT) IN 2 HOURS 12-ii (w) 1 mg/ml 20 5 — 17.18 (Control) 12-jj(b) 1 mg/ml 20 5 0.5 15.81 12-kk (b) 1 mg/ml 20 5 2 10.02 (b)—buffer,(w)—water

TABLE 32 Effect of Tween 80 (TW 80) on Fentanyl Formulations CONC. OFALCO- % EXAMPLE FENTANYL HOL PG TW80 PERMEATED #* BASE % (V) % (V) %(WT) IN 2 HOURS 12-ll(b) 1 mg/ml 20 5 — 17.33 (Control) 12-mm(b) 1 mg/ml20 5 0.3 14.73 12-nn(b) 1 mg/ml 20 5 0.6 13.72 (b)—buffer, (w)—water

TABLE 33 Effect of Labrasol & SLS on Fentanyl Formulations CONC. OF %FENTANYL ALCOHOL PG LABRASOL SLS PERMEATED EXAMPLE #* BASE % (V) % (V) %(WT) % (WT) IN 2 HOURS 12-oo(w) 1 mg/ml 20 5 — — 17.18 (Control)12-pp(w) 1 mg/ml 20 5 6.5 — 8.25 12-qq(w) 1 mg/ml 20 5 3.0 0.5 9.3112-rr(w) 1 mg/ml 20 5 6.5 0.5 8.63 (b)—buffer, (w)—water

TABLE 34 Effect of Mannitol & Triacetin on Fentanyl Formulations CONC.OF % FENTANYL ALCOHOL PG MANNITOL TRIACETIN PERMEATED EXAMPLE #* BASE %(V) % (V) % (WT) % (WT) IN 2 HOURS 12-ss(b) 1 mg/ml 20 5 — — 17.33(Control) 12-tt(b) 1 mg/ml 20 5 0.1 0.15 16.14 12-uu(b) 1 mg/ml 20 50.15 0.4 18.33 12-vv(b) 1 mg/ml 20 5 0.15 0.5 17.14 12-ww(b) 1 mg/ml 205 0.2 0.3 16.73 12-xx(b) 1 mg/ml 20 5 0.25 0.5 16.31 12-yy(b) 1 mg/ml 205 0.3 0.2 17.70 12-zz(b) 1 mg/ml 20 5 0.4 0.2 16.88 (b)—buffer,(w)—water

TABLE 35 Effect of Mannitol & TW 80 on Fentanyl Formulations CONC. OF %FENTANYL ALCOHOL PG % MANNITOL TW80 PERMEATED EXAMPLE #* BASE % (V) (V)% (WT) % (WT) IN 2 HOURS 12-aaa(b) 1 mg/ml 20 5 — — 17.33 (Control)12-bbb(b) 1 mg/ml 20 5 0.2 0.2 18.74 12-ccc(b) 1 mg/ml 20 5 0.3 0.116.53 12-ddd(b) 1 mg/ml 20 5 0.3 0.2 16.02 (b)—buffer, (w)—water

TABLE 36 Effect of Triacetin & TW 80 on Fentanyl Formulations CONC. OF %FENTANYL ALCOHOL PG % TW 80 TRIACETIN PERMEATED EXAMPLE #* BASE % (V)(V) % (WT) % (WT) IN 2 HOURS 12-eee(b) 1 mg/ml 20 5 — — 17.33 (Control)12-fff(b) 1 mg/ml 20 5 0.1 0.2 16.99 12-ggg(b) 1 mg/ml 20 5 0.1 0.316.63 (b)—buffer, (w)—water

The results indicate that addition of individual excipients includingHPBCD (Table 25), PVP (Table 27), PC (Table 28), Triethyl citrate (Table31) and Tween® 80 (Table 32) to the formulation decreased thepermeability of fentanyl across MatTek buccal membranes irrespective ofexcipient concentration. As shown in Table 26 and Table 30, formulationscontaining 0.3% Mannitol and 0.5% Triacetin showed similar permeabilitycharacterisitics as that of control formulation but the permeabilitydecreased as the concentrations of these individual excipients wereincreased in the formulations. Stability studies indicated that aminimum of 0.45% and 0.5% mannitol concentration should be added tobuffer and water formulations, respectively, to keep them stable. Incase of Triacetin, formulations containing 0.5% or higher concentrationsof triacetin were found to be stable.

Permeation of fentanyl from formulations containing SG was greater thanthat of control formulations (Table 29). Also, we observed that fentanylpermeation increased with the increase of SG concentration in theformulation.

The results of fentanyl permeation across MatTek-buccal tissues fromformulations containing combination of excipients are shown in Tables33-36. Addition of Labrasol® (Labrasol is a registered trademark ofGattefosse SAS) to the formulation improved the stability but decreasedfentanyl permeation across MatTek buccal tissues. Similar results wereobserved with formulations containing Labrasol® and SLS. Among all theformulations containing combination of excipients, two formulations,Example 12-uu (0.15% mannitol, 0.4% triacetin) and Example 12-bbb (0.2%mannitol, 0.2% TW80), showed higher permeability compared to controlformulation. We observed that the presence of mannitol in triacetinformulations did not show any improvement in the permeation. Hence, theformulation containing 0.5% triacetin (Table 30) was selected forfurther studies. Though the formulation, Example 12-bbb, showed goodpermeability mannitol concentration was increased to 0.3% to improve thestability of the product.

Both fentanyl citrate and fentanyl base formulations were stable at alltemperatures studied. The data from in-vitro tissue permeation studies,as shown in Table 3, showed that permeation of fentanyl from fentanylbase formulations was about 10-fold higher than from fentanyl citrateformulations. Water and buffer formulations did not show any significantdifference in fentanyl base permeation across buccal tissue. Our studiesalso showed that fentanyl base formulation containing Miglyol® had verylow permeability. Among the excipients, triacetin at 0.5% and mannitolat 0.3% in combination with 0.2% TW 80 showed good permeability andstability.

The transport of fentanyl across buccal tissues was dependent on ethylalcohol (ethanol) and propylene glycol (PG) concentrations in theformulation. Ethanol is used as a cosolvent to solubilize fentanyl basein aqueous based formulations. In this investigation, we observed thatformulations (with 1 mg/ml of fentanyl base) containing less than 20% ofethanol were precipitating at refrigerated conditions. Also, we observedthat permeation of fentanyl across buccal tissue was indirectlyproportional to ethanol concentrations in the formulations (Table 21).Similar results were observed with PG (Table 22). PG was helpful inincreasing the solubility of fentanyl base in aqueous solution and alsoin enhancing the permeation of the fentanyl base across buccal tissues.Formulations containing more than 5% PG did not show significantimprovement in the permeation of fentanyl, however, the solutionviscosities increased proportionally with PG concentrations. Theformulations containing more than 5% PG did not show good spraycharacteristics.

Example 13

In Example 13, a formulation having the following ingredients in Table37 was tested. The formulation did not include fentanyl.

TABLE 37 Ingredient Dehydrated Alcohol % (v/v)  30% Propylene glycol %(v/v)   5% Peppermint Oil % (v/v) 0.2% Borate buffer (pH 9) QS HydroxyPropyl Beta 1:2 Cyclodextrin (HPBCD)

The formulation was sprayed using a 0.10 ml multidose nasal spray pumpby Pfeiffer® of America, Princeton, N.J. (Pfeiffer is a registeredtrademark of Ing. Erich Pfeiffer GmbH) and the droplets were measuredusing a Malvern® Mastersizer® S device, by Malvern® Instruments Ltd. Asingle depression of the sublingual spray pump generated a plume whichwas then analyzed for spray particles. The sample size for the dosevolume, spray pattern, and droplet size distribution was 25 sprays.

Droplet Volume

In the droplet volume evaluation, 25 spray samples were evaluated using5 different stroke numbers for each spray sample the following resultswere measured:

Overall mean=100.4 μl

Maximum single actual value=103.2 μl

Lowest single actual value=95.3 μl

Standard deviation=1.1

Range=7.9

Coefficient of variation=1.1%

Spray Pattern

In the spray pattern evaluation, 25 spray samples were evaluated using amanual actuation at 30 mm from the target. The formulation was dyed withmethylene blue and the following spray pattern results were measured:

Small diameter [mm]

min: 35.4

mean: 50.6

max: 62

s: 7.00

largest diameter [mm]

min: 40

mean: 56.9

max: 67

s: 6.01

spray angle

min: 64°

mean: 83.3°

max: 94°

s: 7.03

ratio (largest/smallest diameter)

min: 1.04

mean: 1.13

max: 1.33

s: 0.073

Droplet Size Distribution

In the droplet size distribution evaluation, 25 spray samples wereevaluated using a manual actuation at 30 mm from the target. Thefollowing droplet size distribution results were measured:

Percentage share of droplet diameters at 10 μm [%]

min: 0.65

mean: 1.66

max: 2.70

s: 0.527

10% of the droplet diameters are smaller than the indicated value [μm]

min: 15

mean: 18.2

max: 23

s: 1.91

50% of the droplet diameters are smaller than the indicated value [μm]

min: 35

mean: 44.7

max: 65

s: 7.52

90% of the droplet diameters are smaller than the indicated value [μm]

min: 96

mean: 154.4

max: 349

s: 64.42

Example 14

In Example 14, a formulation having the following ingredients in Table38 was tested. The formulation did not include fentanyl.

TABLE 38 Ingredient Dehydrated Alcohol % (v/v)  30% Propylene glycol %(v/v)   5% Peppermint Oil % (v/v) 0.2% Borate buffer (pH 9) QS HydroxyPropyl Beta Cyclodextrin 1:2 (HPBCD) Hydroxy Propyl Cellulose (HPC EP)  1% % (v/v)

The formulation was sprayed using a 0.10 ml multidose nasal spray pumpby Pfeiffer® of America, Princeton, N.J. and the droplets were measuredusing a Malvern® Mastersizer® S device, by Malvern® Instruments Ltd. Asingle depression of the sublingual spray pump generated a plume whichwas then analyzed for spray particles. The sample size for the dosevolume, spray pattern, and droplet size distribution was 25 sprays.

Droplet Volume

In the droplet volume evaluation, 25 spray samples were evaluated using5 different stroke numbers the following results were measured:

Overall mean value=101.5 μl

Maximum single actual value=103.7 μl

Lowest single actual value=96.1 μl

Standard deviation=1.3

Range=7.6

Coefficient of variation=1.2%

Spray Pattern

In the spray pattern evaluation, 25 spray samples were evaluated using amanual actuation at 30 mm from the target. The formulation was dyed withmethylene blue and the following spray pattern results were measured:

Small diameter [mm]

min: 29

mean: 34.0

max: 46

s: 3.47

largest diameter

min: 35

mean: 40.8

max: 55

s: 4.13

spray angle

min: 58°

mean: 63.8°

max: 80°

s: 4.65

ratio (largest/smallest diameter)

min: 1.08

mean: 1.20

max: 1.50

s: 0.092

Droplet Size Distribution

In the droplet size distribution evaluation, 25 spray samples wereevaluated using a manual actuation at 30 mm from the target. Thefollowing droplet size results were measured:

Percentage share of droplet diameters at 10 μm [%]

min: 0.24

mean: 0.68

max: 1.28

s: 0.278

10% of the droplet diameters are smaller than the indicated value [μm]

min: 22

mean: 26.7

max: 35

s: 2.95

50% of the droplet diameters are smaller than the indicated value [μm]

min: 61

mean: 83.3

max: 114

s: 11.41

90% of the droplet diameters are smaller than the indicated value [μm]

min: 192

mean: 294.6

max: 440

s: 53.32

Example 15

In Example 15, an assay procedure for fentanyl in 1, 2, 4, 6, and 8mg/mL fentanyl sublingual spray samples with a working concentration ofbetween 0.1 μg/mL and 5 μg/mL fentanyl in solution was performed. Thismethod was developed and qualified in compliance with GMP requirements.The method was determined to be linear over the range of 0.05 μg/mL to7.83 μg/mL fentanyl. The fentanyl working standard solution was stableover a seven day period in volumetric glassware and amber HPLC vials atrefrigerated and ambient conditions.

The equipment and supplies utilized in this process included an HPLCsystem equipped with a pump, variable wavelength detector, andautosampler, or equivalent, a Waters Symmetry HPLC column (C18, 4.6×75mm, 3.5 μm particle size), 0.45 μm, 47 mm nylon filters (Gelman Nylaflo®P/N 66608 or equivalent; Nylaflo is a registered trademark of MembranaInc.), acetonitrile (HPLC Grade), potassium phosphate monobasic (ACSGrade), phosphoric acid (ACS Grade), deionized water, alcohol (ethanol,absolute), and fentanyl base reference standard.

The solution preparations were prepared as described below and may bescaled as required.

For each liter of phosphate buffer solution (50 mM KH₂PO₄ pH 2.8), 6.8 gof potassium phosphate mono basic and 1 liter of water was combined in asuitable vessel and mixed well. The pH of the solution was adjusted to2.8 with the drop-wise addition of phosphoric acid. The solution wasfiltered through 0.45 μm nylon. This solution expires after one month.

For each liter of mobile phase (25% ACN, 75% phosphate buffer) solution,750 mL of phosphate buffer solution was combined with 250 mL ofacetonitrile in a suitable vessel and mixed well. The system is degassedby an appropriate method before use if required. This solution expiresafter one month.

For each liter of stock diluent (95/5, Ethanol/Acetonitrile) solution,950 mL ethanol and 50 mL acetonitrile was combined in a suitablecontainer and mixed well. This solution expires after one month.

Stock standard I, 40 mg/mL, (“SSI”), was prepared by weighingapproximately 10.0 mg of fentanyl reference standard and added to a 250mL volumetric flask. Approximately 200 mL stock diluent was added andswirled to dissolve the solid material. The mixture was diluted to thedesired volume with stock diluent and mixed well.

Stock standard II, 40 μg/mL, (“SSII”), was prepared by weighingapproximately 10.0 mg of fentanyl reference standard and add to a 250 mLvolumetric flask. Approximately 200 mL of stock diluent was added andswirled to dissolve solid material. The mixture was diluted to thedesired volume with stock diluent and mixed well.

Working standard I, 2.4 μg/mL, (“WSI”), was prepared by transferring 3.0mL of stock standard I to a 50 mL volumetric flask. The mixture wasdiluted to the desired volume with mobile phase solution and mixed well.This solution expires after seven days.

Working standard II, 2.4 μg/mL, (“WSII”) was prepared by transferring3.0 mL of stock standard II to a 50 mL volumetric flask. The mixture wasdiluted to the desired volume with mobile phase solution and mixed well.This solution expires after seven days.

The chromatographic conditions for the HPLC procedure are set forthbelow:

-   Column: HPLC Column Waters Symmetry C18, 4.6×75 mm, 3.5 μm particle    size-   UV Detection: 214 nm-   Flow Rate: 2.0 mL/minute-   Injection Volume: 50 μL-   Temperature: Ambient (The temperature may be controlled at 25° C.).-   Acquisition Time: 13 minutes    HPLC Procedure

After the system suitability is established, a maximum of 12 samplesolutions can be injected in between working standards. A typicalsequence would be as follows:

2× Mobile Phase (Blank)

2× Working standard II

5× Working standard I

I× Sample (up to 12 injections)

I× Working standard I

I× Sample (up to 12 injections)

I× Working standard I

System Suitability

There should be no significant interfering peaks present at theretention time of fentanyl in the mobile phase blank injections. Interms of injection precision, the RSD of fentanyl for five replicateinjections of working standard I should not exceed 2.0%. In terms ofstandard agreement, the agreement between the average peak response forthe first five working standard I injections and the two workingstandard II injections should be between 98 to 102%. The agreementbetween working standards I and II need only be demonstrated once duringthe expiry of standards. The tailing factor at 5% peak height forfentanyl in the first working standard I injection should be between 0.8and 1.5. In terms of standard precision over the run, the RSD of peakarea for fentanyl in the working standard I injections over the run(OTR) should not exceed 2.0%.

Calculations are performed as set forth below.

Working standard concentration is calculated as follows:

${\frac{{Mass}\mspace{14mu}{of}\mspace{14mu}{standard}\mspace{14mu}({mg})}{250.0\mspace{14mu}{mL}}*\frac{1000\mspace{14mu}{\mu g}}{1\mspace{14mu}{mg}}*\frac{3.0\mspace{14mu}{mL}}{50.0\mspace{14mu}{mL}}*{Purity}\mspace{14mu}{of}\mspace{14mu}{Standard}} = {{\mu g}\text{/}{mL}\mspace{14mu}{Fentanyl}}$

Response Factor (R_(f)) is calculated as follows:

$\frac{{Fentanyl}\mspace{14mu}{Peak}\mspace{14mu}{Area}}{{Fentanyl}\mspace{14mu}{Concentration}\mspace{14mu}( {{\mu g}\text{/}{mL}} )}*=R_{f}$

Standard Agreement is calculated as follows:

${\frac{{Average}\mspace{14mu} R_{f}{WSII}}{{Average}\mspace{14mu} R_{f}{WSI}}*100} = {\%\mspace{14mu}{Standard}\mspace{14mu}{Agreement}}$

Example 16

In Example 16 the method for determination of droplet size distributionby laser diffraction for fentanyl sublingual spray using the Spraytecdevice by Malvern® was performed.

All data generated and described within this report were reviewed forcompliance with Good Manufacturing Practices (21 CFR Parts 210 and 211).

The purpose of this project was to develop and validate a droplet sizedistribution method by laser diffraction for use with fentanylsublingual spray product and placebo. The first portion of the projectperformed product evaluations to determine the proper automatedactuation parameters to be used with the MightyRunt Actuation Station byInnova Systems, Inc. Using the automated actuation station, developmentof the droplet size distribution method for the sublingual productincluded vignetting studies, exhaust placement studies, and deviceplacement studies.

The method validation evaluated intermediate precision between twoanalysts performing the developed method. All method development andqualification activities were performed using placebo.

Samples were prepared using Pfeiffer® unit dose glass vials, Pfeiffer®unit dose VI stoppers, Pfeiffer® vial holder, and Pfeiffer® unit doseapplicator. The instrumentation utilized in the study include a Spraytecwith 200 mm lens by Malvern® Instruments, Inc, a MightyRunt ActuationStation by Innova Systems Inc. equipped with an exhaust fan attachment,and a Mettler Toledo® balance Model AT201.

Actuation Parameter Study

Using the Spraytec to track the plume duration and droplet sizedistribution, the actuation parameters for the MightyRunt ActuationStation (MightyRunt) were optimized to replicate the plume duration,droplet size distribution, and shot weight generated by manualactuation. Dv10 (10% of the droplet diameters are smaller that theindicated value), Dv50 (50% of the droplet diameters are smaller thatthe indicated value), and Dv90, (90% of the droplet diameters aresmaller that the indicated value), results from six devices with manualactuations were compared with the results from the six devices withautomated actuations.

Acceptance Criteria

The individual shot weight results for the automated actuations shouldall fall within the range of 75%-125% of the average shot weight for themanual actuations. The average Dv10, Dv50 and Dv90 results of theautomated actuations should be within 75% to 125% of the average Dv10,Dv50 and Dv90 results for the manual actuations.

Statistical analysis shall include performance of a students' t-test onthe two sets of droplet size distribution results. The results of thestudents' t-test should indicate that the manual versus automated setsof data is statistically equivalent.

Method Development

Method development involved a vignetting study and exhaust studyutilizing the Spraytec. Actuations were performed using the MightyRuntand previously determined parameters. Vignetting occurs during laserdiffraction analysis when the small droplets of a spray plume scatterthe laser at an angle too steep to be captured by the range lens.Placement of the device close enough to the range lens to capture all ofthe scattered light without deposition on the range lens is critical.The vignetting study determined the appropriate range lens for analysisand the appropriate distance from device to range lens.

Exhaust placement affects plume travel. Plume velocity should not beaccelerated by the draw of the exhaust and large droplets should notfall while traveling through the laser path. The exhaust studydetermined the appropriate position behind the plume of the spray toensure proper plume capture after passing through the laser path. Themethod included two distances for analysis from the tip of the nozzle tothe path of the laser for a more complete characterization of thedroplet size distribution.

Method Validation

Validation consisted of determining the precision and ruggedness of themethod. A total of 24 devices from a single lot of placebo were used inthe validation. One analyst tested 6 actuations at each distance. Todemonstrate ruggedness, a second analyst repeated the analyses. Dv10,Dv50, and Dv90 results were compared.

Acceptance Criteria

The individual shot weight results for analyst one should all fallwithin the range of 75%-125% of the average shot weight for analyst two.The average Dv10, Dv50 and Dv90 results for analyst one should be within75% to 125% of the average Dv10, Dv50 and Dv90 results for analyst two.

The final experimental procedure is set forth below.

Prior to analysis, the background and scattering profiles were verifiedas appropriate for analysis.

The sample bottle is inserted into the nozzle holder. Coasters were usedto raise the platform for minimal adjustment. The coaster attached tothe device holder was placed on top of coasters used for adjustment.

The MightyRunt with bottle and exhaust fan was placed in the appropriatepositions for analysis. The MightyRunt was raised with the nozzlecentered in front of the laser path with a lab jack. It was ensured thatthe MightyRunt was level following adjustment. Two bottle-to-laserdistances were be evaluated, 7 cm and 4 cm, measured from the pump tipto the center of the laser path. For all analyses, the bottle was placed14 cm from the range lens support structure to the pump tip.

The exhaust fan was turned on and placed on a lab jack 3 cm behind thelaser path, centered behind the device, measured from the center of thelaser path to the front edge of the exhaust shield. The exhaust fan hadan impaction surface for the droplets to adhere to, i.e. a cheeseclothplaced in the path of the droplets. ⅛ sheet of 18×36 inch cheeseclothfolded into approximately a 4.5-inch square (4 layers of cheesecloth) isa sufficient impaction surface and will not impede the flow of the fan.

The device was actuated using the MightyRunt station and droplet sizedistribution was collected for each individual shot at the appropriatedistances for analysis.

A report was printed for the entire plume duration and the plumeplateau.

Data Reporting

The data for the D10, D50, D90, and Span values for each actuation arereported. The average and precision (% RSD) (percent relative standarddeviation) were calculated for the D10, D50, and D90. Note: Span isdefined as (D90-D10)/D50. Individual droplet size distribution results(μm) and span values (unitless) were reported to X.XX. All averagedroplet size distribution results should be reported to X.X μm. All RSDvalues should be reported to X.X %.

Statistical analysis included performance of a students' t-test on thetwo sets of droplet size distribution results. The results of thestudents' t-test should have indicated that the analysts sets of data isstatistically equivalent.

Results and Discussion

All spray plumes have three stages. The first stage, development, wascharacterized by variable droplets and decreasing transmission of thelaser. The second stage, stable, was characterized by a stable dropletdistribution and transmission. Variable droplets and increasingtransmission characterize the final stage, dissipation. All comparisonsof droplet size distribution used the stable stage of the plume.

Actuation Parameter Study

During the optimization of the actuation parameters for the MightyRunt,the plumes from manual and automated actuations were measured using theSpraytec. Dv10, Dv50, and Dv90 results were compared to optimize theautomated parameters. The type of device that was examined in this studywas significantly different from typical nasal sprays. Rather than aspring requiring a consistent force to initiate actuation and deliverthe drug, there was an amount of force that was required to break thetabs or actuate the device. This force was not the same amount of forcerequired to deliver the drug from the device once the tabs are broken.Because this device did not contain a spring, the parameters of interestwere actuation force, force rise time, and minimum travel distance. Thefinalized MightyRunt parameters are listed below in Table 39 and theutilized Spraytec settings are listed below in Table 40.

TABLE 39 Mighty Runt Actuation Parameters Parameter Setting ActuationForce 4.0 kg Force Rise Time 0.2 sec Hold Time 1.0 sec Force Fall Time1.0 sec Spray Delay 1 sec Minimum Travel Distance 10 mm Maximum travelTime 4.0 sec Trigger Signal Delay 0.0 sec Stage Yes

TABLE 40 Spraytec Settings Option Setting Test Duration 200 msec DataAcquisition Rate 1000 Hz Acquisition Duty Cycle  0% Experimental TriggerTransmission Transmission Trigger 98% Range Lens 200 mm

The comparison of manual and automated actuations was performed usingsix devices with manual actuations and six devices with automatedactuations. The droplet size distribution and shot weight results aresummarized in Table 41 below.

TABLE 41 Shot Weight % Device D10 (μm) D50 (μm) D90 (μm) (mg) AverageManual 1 19.0 44.5 76.8 89.1 Manual 2 18.1 42.7 84.9 86.9 Manual 3 33.358.7 108.4 90.4 Manual 4 20.8 44.6 81.2 87.0 Manual 5 18.3 42.7 80.787.8 Manual 6 19.0 44.3 81.5 84.9 Average 21.4 46.3 85.6 87.7 % RSD 27.513.3 13.4 2.2 Mighty Runt 1 17.3 37.5 65.1 83.8 96 Mighty Runt 2 16.135.3 64.0 66.0 75 Mighty Runt 3 18.8 38.9 66.2 84.2 96 Mighty Runt 415.4 37.8 77.6 85.4 97 Mighty Runt 5 17.6 38.6 67.1 82.4 94 Mighty Runt6 19.4 41.9 74.8 88.0 100 Average 17.4 38.3 69.1 81.6 % RSD 8.8 5.6 8.29.7 Overall 19.43 42.30 77.36 84.7 average Overall % RSD 23.7 14.3 15.77.5 Automated 81 83 81 Actuation Average as % of Manual ActuationAverage t-stat 1.60 2.97 3.15 t-critical 1.94 1.94 1.81 Result SameDifferent Different population population population

Acceptance Criteria

Shot weight results for the automated actuations ranged from 75%-100% ofthe average for manual actuations, meeting the acceptance criteria of75%-125% of the average shot weight for the manual actuations. Theaverage Dv10 for automated actuations was 81% of the average Dv10 formanual actuations. The average Dv50 for automated actuations was 83% ofthe average Dv50 for manual actuations. The average Dv90 for automatedactuations was 81% of the average Dv90 for manual actuations. Each ofthese parameters met acceptance criteria of 75%-125% of the average formanual actuations. Students' t-tests, while not necessarily appropriatefor the small data sets, indicated that the data sets for manualactuations and automated actuations were equivalent for Dv10, but notfor Dv50 or Dv90. It was not possible to accurately replicate thedroplet size distribution from manual actuations with the MightyRunt.The force required to reliably break the tabs and actuate the devicesproduced a distribution with smaller droplets than that of the manualactuations. Less aggressive actuation parameters, which should producelarger droplet sizes, were not sufficient for consistent actuation ofthe devices. Spraytec method development proceeded with these parametersdespite not meeting the acceptance criteria specified in the protocol.

Method Development

Method development involved a vignetting study, device placement studyand exhaust placement study utilizing the Spraytec. Actuations wereperformed using the MightyRunt and the previously qualified parameters.For all tests, the device was placed in front of the laser beam path,the plume traveled through the laser path, and the plume was collectedin an exhaust manifold placed behind the laser beam.

The vignetting experiments were performed with the device aligned withthe front of the instrument (approximately 10 cm from the beam) andvarying distances for the device-to-range lens placement. The resultsare summarized in Table 42.

TABLE 42 Distance to Range Dv10 Lens (cm) (μm) Dv50 (μm) Dv90 (μm) 617.2 38.6 65.9 16.1 36.5 63.0 8 18.6 41.3 71.6 21.9 44.2 84.6 10 16.738.7 80.4 16.9 39.8 68.0 12 22.1 46.1 89.8 17.1 37.1 65.5 15.6 35.7 62.714 17.4 42.3 72.5 16.6 36.5 64.2

A plot of the Dv10, Dv50, Dv90, and plume records values versusplacement is set forth in FIG. 11. The data showed no significant trendover the entire range of placements. To minimize the possibility ofdeposition of droplets on the range lens during testing, the placementof 14 cm (approximately centered between the laser and range lenssupports) was chosen and used for all further testing.

To evaluate exhaust placement on the plume during testing, PSD (particlesize diameter) data was collected and evaluated at four distances forexhaust-to-laser beam. The data are summarized in Table 43 below.

TABLE 43 Exhaust Height (cm) Dv10 (μm) Dv50 (μm) Dv90 (μm) 7 cmDevice-to-Laser Beam Placement No exhaust 14.4 28.1 51.7 13.3 28.6 54.03 19.8 37.1 60.9 18.1 36.3 61.0 4 cm Device-to-Laser Beam Placement Noexhaust 17.8 35.0 57.9 18.7 36.3 60.5 1 14.6 29.4 51.0 12.6 28.0 56.6 315.1 30.0 52.0 13.6 27.6 49.0 5 14.3 32.8 79.8 13.4 28.7 51.7 7 11.225.5 55.7 15.9 30.0 50.8

A graphical summary of Dv10, Dv50, and Dv90 values versus placement isincluded in FIG. 12. There were no significant changes in the Dv10,Dv50, Dv90 or plume duration values for plumes collected withoutexhaust, or with exhaust 1, 3, 5, or 7 cm behind the beam. Theexhaust-behind-laser beam placement was chosen to be 3 cm to reduce thechance of deposition outside the fan housing of sprays performed at adevice-to-beam distance of 7 cm.

The method was to include two distances for analysis from the tip of thedevice to the path of the laser for a more complete characterization ofthe droplet size distribution. Four distances were evaluated, andresults are included in Table 44 below.

TABLE 44 Distance (cm) Dv10 (μm) Dv50 (μm) Dv90 (μm) 7 16.7 34.5 67.815.8 33.4 60.0 6 17.8 35.0 57.9 18.7 36.3 60.5 5 19.1 35.5 58.5 16.932.4 55.1 4 11.8 27.8 57.5 13.0 28.1 50.3 3 14.4 28.1 51.7 13.3 28.654.0

A graphical summary of Dv10, Dv50, and Dv90 values versus placement areincluded in FIG. 3. There was no observable trend in the data fromvarious heights. While there is no guidance document for sublingualsprays, the FDA Guidance Document “Bioequivalence and BioavailabilityStudies for Nasal Aerosols and Nasal Sprays for Local Action”, specifiestwo distances from 3-7 cm. The larger pair of distances (4 cm and 7 cm)from the beam was chosen in order to characterize a more fully developedplume.

Method Validation

An analyst tested six devices at both the 4 cm and 7 cm distances. Asecond analyst repeated the analyses at both distances with the nextactuations after priming from each device on a second day. Validationresults are summarized in Tables 45 and 46 below where the Dv10, Dv50,and Dv90 results were compared.

TABLE 45 Validation PSD Results at 4 cm Device-to-Laser Beam PlacementShot Weight % Device Dv10 (μm) Dv50 (μm) Dv90 (μm) (mg) Average Analyst1 1 18.9 31.2 52.3 60.4 76 2 16.4 30.8 52.6 78.8 100 3 17.2 29.8 51.365.0 82 4 18.3 31.6 54.2 80.1 101 5 13.5 28.5 51.7 81.6 103 6 15.0 30.152.3 77.2 98 Average 16.5 30.3 52.4 73.9 % RSD 12.3 3.7 1.9 12.0 Analyst2 1 14.2 28.8 57.5 76.8 2 11.3 26.8 54.9 76.9 3 11.8 27.2 52.2 79.9 415.4 29.2 52.6 80.0 5 12.3 27.8 60.2 76.9 6 11.8 26.8 52.2 84.5 Average12.8 27.8 54.9 79.2 % RSD 12.8 3.7 6.0 3.8 Overall 14.7 29.0 53.7 76.5Average Overall 17.9 5.8 5.0 9.0 % RSD Analyst 1 129 109 95 Average as %of Analyst 2 t-stat 3.50 4.20 −1.80 t-critical 1.81 1.81 1.81 ResultDifferent Different Same Population Population Population

TABLE 46 Validation PSD Results at 7 cm Device-to-Laser Beam PlacementDv10 Dv90 ShotWeight % File (μm) Dv50 (μm) (μm) (mg) Average Analyst 1-7cm a009 23.3 37.0 58.8 80.1 104 a010 16.1 34.0 57.3 76.9 99 a011 16.034.9 59.7 79.0 102 a012 17.6 34.0 57.1 80.7 104 a013 20.0 35.5 58.0 82.3106 a014 19.6 36.1 57.2 80.6 104 Average 18.8 35.3 58.0 79.9 % RSD 14.83.4 1.8 2.3 Analyst 2-7 cm b009 11.8 28.7 55.0 72.6 b010 17.0 33.9 61.779.8 b011 15.3 31.8 55.8 74.8 b012 13.8 31.1 54.6 79.7 b013 13.3 30.755.1 80.8 b015 16.6 32.8 56.7 76.3 Average 14.6 31.5 56.5 77.3 % RSD13.8 5.8 4.7 4.2 Overall 16.7 33.4 57.2 78.6 Average Overall % 18.9 7.33.6 3.6 RSD Analyst 1 128 112 103 Average as % of Analyst 2 Averaget-stat 2.93 4.25 1.32 t-critical 1.81 1.83 1.81 Result DifferentDifferent Same Population Population Population

Acceptance Criteria

Analyst 1 shot weight results ranged from 76%-103% of analyst 2 averagefor the 4 cm distance, and 99%-106% for the 7 cm distance, meeting theacceptance criteria of 75%-125%. The average Dv10 for Analyst 1 was 129%of Analyst 2 for the 4 cm distance and 128% for the 7 cm distance. Theaverage Dv50 for Analyst 1 was 109% of Analyst 2 for the 4 cm distanceand 112% for the 7 cm distance. The average Dv90 Analyst 1 was 95% ofAnalyst 2 for the 4 cm distance and 103% for the 7 cm distance. WhileDv10 did not meet acceptance criteria of 75%-125%, this is a measurementof the smallest droplets in the plume and a higher variability is notunexpected. Results for Dv50 and Dv90 met acceptance criteria of75-125%. Students' t-tests, while not necessarily appropriate for thesmall data sets, indicated that the data sets for Analyst 1 and Analyst2 were equivalent for Dv90 at both distances, but not for Dv10 or Dv50at either distance.

Conclusion

A method for the droplet size distribution analysis by laser diffractionfor use with fentanyl sublingual spray was developed and subsequentlyqualified. Acceptance criteria based on statistical analysis withstudents' t-test were deemed inappropriate for the small data sets.Method validation acceptance criteria for agreement of Analysts 1 and 2were determined to be too narrow for the high variability associatedwith measurement of the smallest droplets in the plume (Dv10). Whilethese criteria were not met, the method was deemed acceptable for use.The method is suitable for use within the operating parameters specifiedherein.

Example 17

In Example 17, a study was performed to determine the respirable doseless than 9 μm for fentanyl in 1, 2, 4, 6, and 8 mg/mL fentanylsublingual spray samples with a working concentration of between 0.1μg/mL and 5 μg/mL fentanyl in solution. The method used was qualified incompliance with GMP requirements. The sample solutions were determinedto be stable over a seven-day period in volumetric glassware and amberHPLC vials at refrigerated and ambient conditions.

The HPLC process was consistent with the process described in Example 15above. The materials and supplies utilized in the study includedacetonitrile (HPLC Grade), potassium phosphate monobasic (ACS Grade),phosphoric acid (ACS Grade), deionized water, alcohol (ethanol,absolute), Short Stack Andersen Cascade Impactor set-up consisting of a5-liter expansion chamber, induction port, stages 0, 1, 2, and afterfilter, a vacuum source, in-line flow meter (Sierra Top-Track orequivalent), VWR® (VWR is a registered trademark of VWR International,Inc.) Sterile sampling bags, glass fiber filter, 8.1 cm, externalcalibrated flow meter (Dry-Cal Flow Meter or equivalent), and apneumatic actuator (Innova Systems Mighty Runt or equivalent).

Solution Preparation

Solution preparations were prepared according to the methods describedin Example 15 above and may be scaled as required.

The extraction solution was 50:50 (95/5, Ethanol/Acetonitrile:Water).For every liter of prepared solution, 475 mL ethanol, 25 mL acetonitrileand 500 mL of water was combined in a suitable container and mixed well.This solution expires after one month.

The phosphate buffer solution was prepared in a concentration of 50 mMKH₂PO₄ with a pH 2.8. For every liter of prepared solution, 6.8 gpotassium phosphate mono basic and 1 liter of water is combined in asuitable vessel and mixed well. The pH of the solution was adjusted topH 2.8 with the drop-wise addition of phosphoric acid. The solution wasfiltered through 0.45 μm nylon. This solution expires after one month.

The impactor set-up will consist of a 5-liter expansion chamber,induction port, stages 0, 1, 2, and filter prepared according to thefollowing procedure.

The filter stage was placed onto the impactor base.

An 8.1 cm glass fiber filter was placed into the after filter stage andsecured with a clean rubber o-ring.

A solid plate was placed on top of the filter stage and then stage 2 wasplaced in position.

A plate with center hole cutout was placed on top of stage 2 and thenstage 1 was placed in position.

A plate with center hole cutout was placed on top of stage 1 and thenstage 0 was in position.

The cone was placed in position and the impactor was secured with thehold down clamps.

The induction port was affixed to the cone and the 5 L expansion chamberwas placed on top of the induction port.

Testing Set-Up

The testing instrumentation was set up by placing an in-line flow meterbetween the vacuum source and cascade impactor with appropriate tubing.A leak test was performed on the impactor. A flow through the impactorwas started by opening the vacuum source and the flow was adjusted toapproximately 28.3 L/min. A hand was placed over the spray actuationport on the expansion chamber. The flow rate as indicated on the in-linemeter was expected to fall to zero. If a flow was still registered, thecondition of the impactor a-rings was checked, and the test repeated. Toset the flow rate, the expansion chamber was removed from the inductionport and an external calibrated flow meter was attached to the inductionport and the flow was started. The flow was adjusted to 28.3±1 L/minwith the external calibrated meter and the measurement displayed on thein-line flow meter was recorded for using during the testing procedure.

Testing Procedure

Two devices were actuated for the 1 mg/mL product strength for eachassay result. One device was actuated for the 2, 4, 6, and 8 mg/mLproduct strength. With the expansion chamber in place, the vacuum wasstarted and the flow adjusted to the measurement obtained in during thetesting set-up.

The pre-actuated weight of the device in grams to a minimum of 4 decimalplaces was recorded. The device was positioned so that the spray wouldtravel directly toward the wall opposite the actuation port. Thesublingual spray device was actuated into the expansion chamber with theautomated pneumatic actuator method parameters listed in Table 47.

TABLE 47 Parameter Setting Actuation Force (kg) 5.0 Force Rise Time (s)0.1 Hold Time (s) 1.0 Force Fall Time (s) 1.0 Spray Delays (s) 1 MinimumTravel Distance (mm) 10.0 Maximum Travel Time (s) 4.0 Trigger SignalDelay (s) 0.0 Stage Yes

The post actuated weight of the device in grams is recorded to a minimumof 4 decimal places.

Extraction Procedure

Extraction was accomplished by breaking down the impactor set-up andextracting each component by the following procedures:

The plates and the filter were each extracted separately in bags with10.0 mL of extraction solution. The sample was extracted by hand shakingand kneading for at least one minute. 6.0 mL of the extracted sample wastransferred to a 10 mL volumetric flask and diluted to volume withphosphate buffer solution and mixed well. This is the sample solution.This solution expires after seven days.

The expansion chamber is inverted and place in a holder. The inductionport and cone is inserted into the expansion chamber ground glass joint.Approximately 40 mL of extraction solution is rinsed through the coneand induction port into the expansion chamber. The solution is swirledin the expansion chamber in an effort to extract the entire interiorsurface. The solution is decanted into a 200 mL volumetric flask. Therinse is repeated two additional times for a total rinse volume ofapproximately 120 mL. The flask is brought to volume with phosphatebuffer solution and mixed well. This is the sample solution. Thissolution expires after seven days.

Assay

The sample solutions are assayed as per the procedure set forth inExample 15 above. Calculations are performed as follows.

${{Response}\mspace{14mu}{Factor}\mspace{14mu}({Rf})} = \frac{{Fentanyl}\mspace{14mu}{Peak}\mspace{14mu}{Area}}{{Fentanyl}\mspace{14mu}{Concentration}\mspace{11mu}( {{\mu g}/{mL}} )}$

The fentanyl in the plate and filter samples were determined accordingto the following calculation:

${{The}\mspace{14mu}{amount}\mspace{14mu}{of}\mspace{14mu}{fentanyl}\mspace{14mu}{in}\mspace{14mu}{\mu g}} = \frac{A_{samp}*D_{f}}{{WSI}\mspace{14mu} R_{f}{OTR}}$A_(samp)=Area of fentanyl in sample preparationD_(f)=Dilution factor of fentanyl sample solution preparation (10.0mL×10.0 ml/6.0 mL)WSI R_(f) OTR=Working standard I response factor over the run

The fentanyl in the cone, induction port and expansion chamber isdetermined according to the following calculation:

${{Amount}\mspace{14mu}{of}\mspace{14mu}{fentanyl}\mspace{14mu}{in}\mspace{14mu}{\mu g}} = \frac{A_{samp}*V_{samp}}{{WSI}\mspace{14mu} R_{f}{OTR}}$A_(samp)=Area of fentanyl in sample preparationV_(samp)=Volume of fentanyl in sample solution preparation (200 mL)WSI R_(f) OTR=Working standard I response factor over the run

The respirable dose for 2, 4, 6, and 8 mg/mL is calculated as follows:

Respirable dose in μg=

Sum of the Drug Mass in Particle Size Fraction Less Than 9 μm (μg)

The respirable dose for 1 mg/mL is calculated as follows:

${{Respirable}\mspace{14mu}{dose}\mspace{14mu}({\mu g})} = \frac{\begin{matrix}{{Sum}\mspace{14mu}{of}\mspace{14mu}{the}\mspace{14mu}{drug}\mspace{14mu}{mass}\mspace{14mu}{in}\mspace{14mu}{particle}} \\{{size}\mspace{14mu}{fraction}\mspace{14mu}{less}\mspace{14mu}{than}\mspace{14mu} 9\mspace{14mu}{\mu g}\mspace{14mu}({\mu g})}\end{matrix}}{2( {{number}\mspace{14mu}{of}\mspace{14mu}{actuations}} )}$

The respirable fraction is calculated as follows:

${{Percent}\mspace{14mu}(\%)\mspace{14mu}{Respirable}\mspace{14mu}{fraction}} = {\frac{\begin{matrix}{{Drug}\mspace{14mu}{mass}\mspace{14mu}{in}\mspace{14mu}{particle}\mspace{14mu}{size}\mspace{14mu}{fraction}} \\{{less}\mspace{14mu}{than}\mspace{14mu} 9\mspace{14mu}{\mu m}\mspace{14mu}({\mu g})( {{respirable}\mspace{14mu}{dose}} )}\end{matrix}}{{Total}\mspace{14mu}{Drug}\mspace{14mu}{Mass}\mspace{14mu}({\mu g})} \times 100}$

The particle size cutoff diameters for reporting is set forth in Table48 below.

TABLE 48 Impactor Component Particle Size Grouping Expansion Chamber,Induction Port,  ≧9 μm and Cone Plate 0 Plate 1 9 μm > X ≧ 5.8 μm Plate2 <5.8 μm F

The results for the fentanyl sample assay results in pg, the respirabledose in the particle size fraction less than 9 μm (as per Table 48) inμg, and the percent respirable dose less than 9 μm in percent to onedecimal place were reported.

The Certificate of Analysis for the determination of the respirable 1mg/mL dose set forth in Table 49 below.

TABLE 49 Determination of Respirable Dose, 1 mg/mL Fentanyl Test MethodSpecification Assay of Fentanyl in Described in Example 17 ReportResults sublingual spray samples Determination of respirable ReportResults dose in fentanyl sublingual spray by cascade impaction AverageTotal Fentanyl Particle Shot Mass Respirable CI (μg/ Size Groupingsweight <9 μm dose <9 μm Run Sample dose) groupings percent (mg) (μg)(μg) 1 Globe 76.5694 ≧9 μm 96.4 85.4 2.9 3.6 Plate 0 0.5479 Plate 10.6228 9 μm > X ≧ 0.8 5.8 μm Plate 2 0.4746 <5.8 μm 2.9 Filter 1.8149 2Globe 78.6941 ≧9 μm 96.6 84.0 2.8 3.4 Plate 0 0.6746 Plate 1 0.6217 9μm > X ≧ 0.8 5.8 μm Plate 2 0.5000 <5.8 μm 2.6 Filter 1.6740 3 Globe78.0529 ≧9 μm 97.1 85.3 2.3 2.9 Plate 0 0.5082 Plate 1 0.5429 9 μm > X ≧0.7 5.8 μm Plate 2 0.4185 <5.8 μm 2.2 Filter 1.3596 Average percentrespirable dose 3.3

Example 18 Fentanyl SL Spray Formulations

In Example 18, formulations 18A-18E were prepared in accordance with theprocedures set forth in the above examples. The formulations are setforth in Table 50 below.

TABLE 50 Quantity % w/w Formu- Formu- Formu- Formu- Formu- lation lationlation lation lation Component 18A 18B 18C 18D 18E Fentanyl 0.111 0.2220.4444 0.6667 0.8889 base (1 mg/mL) (2 mg/mL) (4 mg/mL) (6 mg/mL) (8mg/mL) Dehydrated 55.00 55.00 55.00 55.00 55.00 alcohol Propylene 5.005.00 5.00 5.00 5.00 glycol L-Menthol 0.05 0.05 0.05 0.05 0.05 Xylitol3.00 3.00 3.00 3.00 3.00 Purified 36.839 36.728 36.5006 36.2833 36.0611water TOTAL 100 100 100 100 100

Example 19 Clinical Study

A Phase I, single-dose, open-label, randomized, three-period,three-treatment crossover study was conducted to compare the rate ofabsorption and bioavailability of Fentanyl Sublingual Spray 400 mcg toActiq® 400 mcg and to Fentanyl Citrate Injection (IV) 100 mcg.

Subjects received each of the treatments below in randomized fashionduring the three treatment periods, separated by a washout period of atleast 7 days:

-   -   Treatment A: Fentanyl Sublingual Spray (Formulation 18C of        Example 18)        -   Dose=1×400 mcg sublingual spray        -   Insys Therapeutics, Inc.    -   Treatment B: Actiq®        -   Dose=1×400 mcg oral transmucosal unit        -   Cephalon    -   Treatment C: Fentanyl Citrate Injection        -   Dose=1×100 mcg IV        -   Hospira, Inc

Blood samples (1×6 mL) were collected in vacutainer tubes containingEDTA as a preservative from the subjects at 0 (pre-dose) and at 5, 10,20, 30, and 40 minutes after dosing and at 1.0, 1.25, 1.5, 2.0, 4.0,6.0, 8.0, 10.0, 12.0, 16.0, 24.0, and 36.0 hours after dosing. Theplasma samples were then analyzed for fentanyl using a validated liquidchromatography tandem mass spectrometry (LC-MS-MS) procedure. The methodwas validated for a range of 0.0250 to 5.00 ng/mL for fentanyl, based onthe analysis of 0.500 mL of EDTA human plasma.

Data from 21 subjects who completed the study was subjected topharmacokinetic and statistical analyses. Concentration-time data wastransferred from Watson LIMS directly to WinNonlin Enterprise Edition(Version 4.0, Pharsight® Corporation; Pharsight is a registeredtrademark of Tripos L.P.) using the Custom Query Builder option foranalysis. Data was analyzed by noncompartmental methods in WinNonlin.Concentration-time data that were below the limit of quantification(BLQ) were treated as zero (0.00 ng/mL) in the data summarization anddescriptive statistics.

In the pharmacokinetic analysis, BLQ concentrations were treated as zerofrom time-zero up to the time at which the first quantifiableconcentration was observed; embedded and/or terminal BLQ concentrationswere treated as “missing”.

Pharmacokinetic parameters were then calculated for each formulationusing non-compartmental methods. Specifically, the followingpharmacokinetic parameters were calculated: peak concentration in plasma(C_(max)), time to peak concentration (T_(max)), elimination rateconstant (λ_(z)), terminal half-life (T_(1/2)), area under theconcentration-time curve from time-zero to the time of the lastquantifiable concentration (AUC_(last)), and area under the plasmaconcentration time curve from time-zero extrapolated to infinity(AUC_(inf)).

The bioavailability (F) after oral/sublingual administration was alsocalculated. The bioavailability was calculated by using the followingequation:

${F = \frac{{Dose}_{IV}*{AUC}_{extravascualr}}{{Dose}_{extravascular}*{AUC}_{IV}}},$where Dose_(IV) and Dose_(extravascular) are the IV and extravascular(sublingual spray and Actiq®) doses, respectively, and AUC_(IV) andAUC_(extravascular) are the areas under the plasma concentration-timeprofiles after IV and extravascular administration, respectively.Individual AUC_(last) and AUC_(inf) values after each treatment wereused for the calculations.

To compare the fentanyl sublingual spray to Actiq®, analysis of variance(ANOVA) and the Schuirmann's two one-sided t-test procedures at the 5%significance level were applied to the log-transformed pharmacokineticexposure parameters, C_(max), AUC_(last), and AUC_(inf). The 90%confidence intervals for the ratio of the geometric means(Test/Reference) were calculated.

The mean concentration-time data are shown in Table 51 and FIG. 15below. Results of the pharmacokinetic and statistical analyses are shownin Table 52 and Table 53 below. The bioavailability estimates aresummarized in Table 54 below.

TABLE 51 Fentanyl Concentration-Time Data after Administration ofFentanyl Sublinqual Spray 400 mcg (Treatment A), Actiq ® 400 mcg(Treatment B) and Fentanyl Citrate Injection 100 mcg (Treatment C)Treatment A: Treatment B: Treatment C: Fentanyl Sublinqual Spray Actiq ®Fentanyl Citrate Injection 400 mcg 400 mcg 100 mcg Time Mean SD CV MeanSD CV Mean SD CV (hr) n (ng/mL) (ng/mL) (%) n (ng/mL) (ng/mL) (%) n(ng/mL) (ng/mL) (%) 0.00 21 0.00 0.00 NC 21 0.00 0.00 NC 21 0.00 0.00 NC0.08 21 0.168 0.189 112.68 21 0.00146 0.00669 458.26 21 0.749 0.60781.08 0.17 21 0.440 0.300 68.30 21 0.0396 0.0511 129.24 21 0.730 0.24032.84 0.33 21 0.513 0.242 47.24 21 0.220 0.150 68.04 21 0.494 0.18537.44 0.50 21 0.606 0.288 47.45 21 0.324 0.112 34.56 21 0.416 0.13733.01 0.67 21 0.613 0.238 38.81 21 0.360 0.127 35.10 21 0.376 0.10828.64 1.00 21 0.670 0.264 39.32 21 0.444 0.170 38.26 21 0.327 0.077323.61 1.25 21 0.679 0.204 30.11 21 0.478 0.163 34.07 21 0.299 0.068923.04 1.50 21 0.694 0.203 29.19 21 0.533 0.198 37.23 21 0.275 0.060722.05 2.00 21 0.674 0.182 26.98 21 0.570 0.173 30.40 21 0.229 0.047520.74 4.00 21 0.449 0.126 28.05 21 0.363 0.146 40.04 21 0.141 0.033924.00 6.00 21 0.218 0.0793 36.31 21 0.185 0.0713 38.57 21 0.0693 0.016724.16 8.00 21 0.170 0.0673 39.72 21 0.132 0.0519 39.43 21 0.0502 0.015731.19 10.00 21 0.159 0.0720 45.44 21 0.123 0.0609 49.39 21 0.0489 0.020942.68 12.00 21 0.0886 0.0396 44.65 21 0.0710 0.0399 56.20 21 0.01880.0193 102.75 16.00 21 0.0614 0.0322 52.50 21 0.0497 0.0299 60.15 210.00877 0.0145 165.79 24.00 21 0.0484 0.0357 73.79 21 0.0345 0.031992.44 21 0.00777 0.0171 220.03 36.00 21 0.00876 0.0164 187.39 21 0.004650.0123 263.87 21 0.00 0.00 NC Note: Plasma samples analyzed using abioanalytical method with a validated range 0.0250 to 5.00 ng/mL;concentrations below limit of quantification set to zero (0.00 ng/mL) inthe data summarization NC = Not calculated

TABLE 52 Pharmacokinetic Parameters of Fentanyl Treatment A: TreatmentB: Treatment C: Fentanyl Sublinqual Spray Actiq ® Fentanyl CitrateInjection 400 mcg 400 mcg 100 mcg Parameter n Mean SD CV % n Mean SD CV% n Mean SD CV % T_(max) (hr) 21 1.28 0.60 47.18 21 1.70 0.42 25.04 210.16 0.08 50.52 C_(max) 21 0.813 0.252 31.01 21 0.607 0.185 30.55 210.929 0.515 55.48 (ng/mL) AUC_(last) 21 4.863 1.708 35.12 21 3.677 1.44039.17 21 1.688 0.4114 24.38 (hr * ng/mL) AUC_(inf) 16 5.761 1.916 33.2618 4.182 1.670 39.93 16 1.758 0.3822 21.74 (hr * ng/mL) AUC_(Extrap) 1610.26 5.66 55.19 18 10.64 5.68 53.38 16 11.14 3.44 30.92 (%) λ₂ (hr⁻¹)16 0.0904 0.0571 63.16 18 0.1097 0.0532 48.51 16 0.1775 0.0662 37.30T_(1/2) (hr) 16 9.98 4.41 44.14 18 7.89 3.72 47.15 16 4.50 1.94 43.02T_(last) (hr) 21 25.15 7.17 28.50 21 22.86 6.83 29.87 21 13.81 5.5139.90 C_(last) 21 0.0408 0.0117 28.74 21 0.0363 0.00953 26.23 21 0.03520.0117 33.08 (ng/mL)

TABLE 53 Statistical Analysis of the Log-Transformed Systemic ExposureParameters of Fentanyl Comparing Fentanyl Sublingual Spray 400 mcg(Treatment A) to Actiq ® 400 mcg (Treatment B) Dependent GeometricMean^(a) Ratio (%)^(b) 90% CI^(c) ANOVA Variable Test Ref (Test/Ref)Lower Upper Power CV % ln(C_(max)) 0.7865 0.5884 133.67 119.67 149.310.9527 20.85 ln(AUC_(last)) 4.6392 3.4767 133.44 121.47 146.58 0.985917.65 ln(AUC_(inf)) 5.5080 4.0420 136.27 121.21 153.20 0.9341 17.06^(a)Geometric Mean for Treatment A (Test) and Treatment B (Ref) based onLeast Squares Mean of log-transformed parameter values ^(b)Ratio(%) =Geometric Mean (Test)/Geometric Mean (Ref) ^(c)90% Confidence Interval

TABLE 54 Bioavailability of Fentanyl after Administration of FentanylSublingual Spray (Treatment A) and Actiq ® (Treatment B) F(AUC_(last))F(AUC_(inf)) Treatment n Mean SD CV (%) n Mean SD CV (%) A 21 0.7210.199 27.59 11 0.756 0.212 28.02 B 21 0.540 0.135 25.08 13 0.511 0.097319.05

It was concluded that Fentanyl SL intermediates between Actiq® andFentanyl IV, as to time to reach threshold concentration, and thatFentanyl SL concentrations are about 40% higher than Actiq®(Bioavailbility is 71±16% vs. 52±13% for Actiq®). Terminal half-life issimilar in all three treatments (Fentanyl IV has an artifactuallydifferent decline beyond 10 hr because concentrations on many subjectsfell below assay sensitivity (Assay sensitivity=0.050 ng/mL (50 pg/mL)).

It was further concluded, based on reports of time to onset for Actiq®(30 min) and Fentora (15 min), that threshold concentration of fentanylis probably ˜0.3-4 ng/mL. Fentanyl SL reaches 0.3-0.4 ng/mL 10 minutespost dose, which is 20 minutes sooner than Actiq®.

Example 20 Dose Escalation Clinical Study

A dose escalation Phase I study on Fentanyl SL has been initiated.Formulation 18A, Formulation 18B, Formulation 18C, Formulation 18D, andFormulation 18E, all of Example 18 were used. Doses of 100 mcg, 200 mcg,400 mcg, 600 mcg, and 800 mcg were administered to 40, 39, 42, 40, and42 patients, respectively. The bio-analytical method was performedaccording to the procedure set forth in Example 19 above. Thepreliminary pharmacokinetic data is provided in Table 54 below, and isgraphically represented in FIG. 16, FIG. 17 and FIG. 18.

TABLE 54 Dose Adj'ed Dose Adj'ed N Cmax AUC(0-t) Cmax AUC # ng/ml Tmax hng * h/mL (to 100 mg) (to 100 mg) 100 mcg 40 0.193 1.12 0.919 0.1930.919 200 mcg 39 0.379 1.04 1.94 0.189 0.971 400 mcg 42 0.796 0.970 4.580.199 1.15 600 mcg 40 1.15 0.987 6.65 0.192 1.11 800 mcg 42 1.56 1.068.93 0.195 1.12

The preliminary data appears to support the conclusion that Fentanyl SLhas faster onset (60% T_(max) in 10 minutes) and therefore pain reliefthan analgesic products currently available commercially (i.e., Actiq®,Fentora®; fentanyl buccal tablet, Fentora is a registered trademark ofFentora Cima Labs Inc., Rapinyl®; fentanyl citrate, Rapinyl is aregistered trademark of Endo Pharmaceuticals Inc., BEMA Fentanyl). Thepreliminary data also indicates that Fentanyl SL stays close to T_(max)for 100 minutes translating to pain relief for a longer time.

Example 21 Pharmacokinetic Profile of Fentanyl Sublingual (SL) Spray

In Example 21, a five-treatment, five-sequence, five-period crossoverstudy of fentanyl SL spray was conducted under fasted conditions in upto 70 healthy subjects. The objectives were to determine thepharmacokinetics of five difference doses (Part A), and to assess theimpact of temperature and pH in the oral cavity on the relativebioavailability at a fixed dose (Part B). Healthy subjects had to meetpre-specified eligibility criteria. Plasma samples were obtained at timepoints of 0, 5, 10, 20, 30, 40 min, 1, 1.25, 1.5, 2, 4, 6, 8, 10, 12,16, 24 and 36 h post-dose and analyzed for fentanyl using a validatedLC-MS-MS procedure.

53 subjects were enrolled in part A. Administration of fentanyl SL spraywas dose-proportional over the 100 mcg to 800 mcg dose ranges. Fentanylconcentrations increase rapidly following administration, being abovethe LLOQ within 5 minutes, reaching 60.6% of the peak plateau by 10minutes and 86.6% of the peak plateau by 20 minutes post dose. Fentanylconcentrations showed a relatively long plateau about the peak value(>80% of Cmax) that lasted approximately 2 hours.

14 subjects were enrolled in part B. Varying the pH and temp of the oralcavity did not affect the PK profile. No SAEs were noted. AEs wereobserved in 31 subjects in part A. 46 were probably related to studytreatment, and 29 were possibly related. During part B, AEs wereobserved in 9 subjects. 7 were probably related to study treatment, and17 were possibly related. AEs were emesis or nausea.

The results of this study support the rationale for assessing efficacyin patients with breakthrough pain. The dose proportionality supports arationale for predictable dosing favorable for titration.

Example 22 Comparative Bioavailability of Fentanyl Sublingual (SL)Spray, IV Fentanyl Citrate and Actiq

In Example 22, a single-dose, open-label, randomized, three-period,three-treatment crossover study with a washout period of at least sevendays between study periods was conducted at a phase I contract clinicunder good clinical practice guidelines. 40 healthy volunteers wereenrolled, having met pre-specified eligibility criteria. Subjectsreceived a single dose of fentanyl SL spray 400 mcg, Actiq 400 mcglozenge, and fentanyl citrate 100 mcg by IV injection over 5 minutes in3 separate treatment periods. Plasma samples were obtained at timepoints of 0, 5, 10, 20, 30, 40 min, 1, 1.25, 1.5, 2, 4, 6, 8, 10, 12,16, 24 and 36 h post-dose and analyzed for fentanyl using a validatedLC-MS-MS procedure.

The results showed that compared to intravenous administration, themedian value for absolute bioavailability of fentanyl SL spray was60.8%; bioavailability of Actiq was 46.6%. The median value for relativebioavailability of Fentanyl SL Spray to that of Actiq was 135%. Systemicabsorption of fentanyl SL was more rapid than Actiq. Subjects weremonitored for any adverse events. AEs were reported in 15 of the 40subjects. All of the AEs were mild. Two of the AEs were probably relatedto the study drug (both were sublingual burning at 400 mcg). Three ofthe AEs were possibly related to the study treatment (headache,dizziness, and dry throat all reported at 400 mcg).

The results of this study support the rationale for assessing efficacyin patients with breakthrough pain.

Many other variations of the present invention will be apparent to thoseskilled in the art and are meant to be within the scope of the claimsappended hereto, including but not limited to the particular unit doseor bi-dose devices and the particle size range of fentanyl produced, aswell as other numerical parameters described in the examples, and anycombination thereof.

What is claimed is:
 1. A sublingual formulation comprising from about0.001% to about 15% by weight fentanyl, a free base, or apharmaceutically acceptable salt thereof, from about 20% to about 60% byweight ethanol, and from about 4% to about 6% by weight propyleneglycol, the formulation providing a mean T_(max) of about 1.28+/−0.60hours when a dose is administered sublingually to humans.
 2. Asublingual formulation comprising from about 0.001% to about 15% byweight fentanyl, a free base, or a pharmaceutically acceptable saltthereof, from about 50% to about 60% by weight ethanol, and from about4% to about 6% by weight propylene glycol, which provides a plasmaconcentration after administration to humans selected from the groupconsisting of: about 60% of the mean C_(max) in about 10 minutes, about86% of the mean C_(max) by about 20 minutes and a combination thereof.3. The sublingual formulation of claim 1, that when administered tohumans provides a plasma concentration that is greater than about 80% ofthe mean C_(max) for about 2 hours.
 4. A sublingual spray formulationcomprising 400 mcg dose of fentanyl, a free base, or a pharmaceuticallyacceptable salt thereof, which provides one or more mean pharmacokineticvalues selected from the group consisting of: AUC_(last) 4.863+/−1.70821hr*ng/mL, AUC_(inf) 5.761+/−1.916 hr*ng/mL, and AUC_(extrap)10.26+/−5.66%, when administered to humans.
 5. A sublingual sprayformulation comprising a dose of fentanyl, a free base, or apharmaceutically acceptable salt thereof, which provides a substantiallydose proportional mean AUC_(last) based on a mean AUC_(last) of about4.863+/−1.70821 hr*ng/mL for a 400 mcg fentanyl dose when administeredto humans.
 6. A sublingual spray formulation comprising a 400 mcg doseof fentanyl, a free base, or a pharmaceutically acceptable salt thereof,which provides a mean F(AUC_(last)) of about 0.721+/−0.199 ng/mL whenadministered to humans.